Compositions and methods relating to anti-IGF-1 receptor antibodies

ABSTRACT

The present invention provides compositions and methods relating to or derived from anti-IGF-1R antibodies. In particular embodiments, the invention provides fully human, humanized, or chimeric anti-IGF-1R antibodies that bind human IGF-1R, IGF-1R-binding fragments and derivatives of such antibodies, and IGF-1R-binding polypeptides comprising such fragments. Other embodiments provide nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having IGF-1R-related disorders or conditions.

REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 13/891,781,filed May 10, 2013, now U.S. Pat. No. 8,895,008, which is a divisionalof U.S. application Ser. No. 13/432,472, filed Mar. 28, 2012, now U.S.Pat. No. 8,460,662, which is a divisional of U.S. application Ser. No.12/954,518, filed Nov. 24, 2010, now U.S. Pat. No. 8,168,409, which is adivisional of U.S. application Ser. No. 11/313,209, filed Dec. 20, 2005,now U.S. Pat. No. 7,871,611, which claims the benefit of U.S.Provisional Application Ser. No. 60/638,961, filed on Dec. 22, 2004. Theabove-identified applications are incorporated herein by reference.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format via EFS-Web. The Sequence Listing is provided as atext file entitled A954USDIV4st25.txt, created Nov. 4, 2014, which is393,502 bytes in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This application provides compositions and methods relating toanti-IGF-1 receptor antibodies.

BACKGROUND OF THE INVENTION

Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2, respectively)promote the differentiation and proliferation of a wide variety ofmammalian cell types.

IGF-1 and IGF-2 both circulate widely throughout the body in plasma.They exert their effects on cells by binding to and activating the IGF-1receptor (IGF-1R). IGF-1R is a member of the family of tyrosine kinasegrowth factor receptors. Its amino acid sequence is about 70% identicalto that of the insulin receptor.

Abnormal IGF-1, IGF-2, and/or IGF-1R activities are associated with anumber of medical conditions, including various types of cancer, growthdefects (e.g., acromegaly, gigantism, and small stature), psoriasis,atherosclerosis, post angioplasty smooth muscle restonsis of bloodvessels, diabetes, microvasular proliferation, neuropathy, loss ofmuscle mass, and osteoporosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1M provide nucleotide sequences encoding light chain variabledomains L1 through L52 and heavy chain variable domains H1 through H52.

FIGS. 2A-2B provide amino acid sequences of light chain variable domainsL1 through L52. CDR and FR regions are indicated.

FIGS. 3A-3B provides amino acid sequences of heavy chain variabledomains H1 through H52. CDR and FR regions are indicated.

FIG. 4 provides amino acid sequences of the light chain CDR1 regions oflight chain variable domains L1 through L52. Consensus sequences forgroups of related CDR sequences are also provided.

FIG. 5 provides amino acid sequences of the light chain CDR2 regions oflight chain variable domains L1 through L52. Consensus sequences forgroups of related CDR sequences are also provided.

FIG. 6 provides amino acid sequences of the light chain CDR3 regions oflight chain variable domains L1 through L52. Consensus sequences forgroups of related CDR sequences are also provided.

FIG. 7 provides amino acid sequences of the heavy chain CDR1 regions ofheavy chain variable domains H1 through H52. Consensus sequences forgroups of related CDR sequences are also provided.

FIG. 8 provides amino acid sequences of the heavy chain CDR2 regions ofheavy chain variable domains H1 through H52. Consensus sequences forgroups of related CDR sequences are also provided.

FIGS. 9A-9B provide amino acid sequences of the heavy chain CDR3 regionsof heavy chain variable domains H1 through H52. Consensus sequences forgroups of related CDR sequences are also provided.

FIG. 10 provides the amino acid sequence of a human IGF-1R extracellulardomain fused to a human IgG1 Fc region (underlined) with an interveningcaspace-3 cleavage site (bold).

FIG. 11 provides the amino acid sequence of a human insulin receptorextracellular domain fused to a human IgG1 Fc region (underlined).

FIG. 12 provides the protein sequence of a human IGF-1R extracellulardomain (including signal peptide) fused at the C-terminus with chickenavidin. The initiating met in the IGF-1R ECD is designated position 1 inthis figure.

FIG. 13 provides the polypeptide sequence of a human kappa light chainantibody constant region and a human IgG1 heavy chain antibody constantregion.

FIG. 14 provides a graph illustrating that four phage-displayedantibodies bind significantly better to an IGF-1R-Fc molecule than theybind to an insulin-receptor-Fc or a murine Fc.

FIGS. 15A-15B provide graphs illustrating the ability of certainantibodies to compete for binding to IGF-1R with IGF-1 and IGF-2.

FIGS. 16A-16F provide graphs illustrating the ability of certainantibodies to inhibit the growth of 32D hu IGF-1R+IRS-1 cells.

FIGS. 17A-17F provide graphs illustrating the ability of certainantibodies to inhibit the growth of Balb/C 3T3 hu IGF-1R cells.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated antigenbinding protein comprising either: a. a light chain CDR3 comprising asequence selected from the group consisting of: i. a light chain CDR3sequence that differs by no more than a total of two amino acidadditions, substitutions, and/or deletions from a CDR3 sequence selectedfrom the group consisting of the light chain CDR3 sequences of L1-L52 asshown in FIG. 6; ii. M X₁ X₂ X₃ X₄ X₅ P X₆ X₇; ill. Q Q X₈ X₉ X₁₀ X₁₁ PX₁₂ T; and iv. Q S Y X₁₃ X₁₄ X₁₅N X₁₆ X₁₇ X₁₈; b. a heavy chain CDR3comprising a sequence selected from the group consisting of: i. a heavychain CDR3 sequence that differs by no more than a total of three aminoacid additions, substitutions, and/or deletions from a CDR3 sequenceselected from the group consisting of the heavy chain CDR3 sequences ofH1-H52 as shown in FIG. 9; ii. X₁₉ X₂₀ X₂₁ X₂₂ X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ F DI; iii. X₂₈ X₂₉ X₃₀ X₃₁ X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ M D V; iv. D S SX₃₉; or c. the light chain CDR3 sequence of (a) and the heavy chain CDR3sequence of (b); wherein X₁ is a glutamine residue or a glutamateresidue, X₂ is an alanine residue, a glycine residue, a threonineresidue, or a serine residue, X₃ is a leucine residue, a phenylalanineresidue, or a threonine residue, X₄ is glutamine residue, a glutamateresidue, or a histidine residue, X₅ is a threonine residue, a methionineresidue, a tryptophan residue, or a valine residue, X₆ is a glycineresidue, an alanine residue, a valine residue, a leucine residue, anisoleucine residue, a proline residue, a phenylalanine residue, amethionine residue, a tryptophan residue, or a cysteine residue, X₇ isthreonine residue, an alanine residue, or a serine residue, X₈ is anarginine residue, a serine residue, a leucine residue, or an alanineresidue, X₉ is an asparagine residue, a serine residue, or a histidineresidue, X₁₀ is an asparagine residue or a serine residue, X₁₁ is atryptophan residue, a valine residue, a tyrosine residue, a prolineresidue, or a phenylalanine residue, X₁₂ is a leucine residue, atyrosine residue, or an isoleucine residue, X₁₃ is an aspartate residueor a glutamine residue, X₁₄ is a serine residue or a proline residue,X₁₅ is a serine residue, a tyrosine residue, an aspartate residue, or analanine residue, X₁₆ is a glutamine residue, an arginine residue, avaline residue, or a tryptophan residue, X₁₇ is an arginine residue, avaline residue, an isoleucine residue, or no residue, X₁₈ is a valineresidue or no residue, X₁₉ is a glutamate residue or no residue, X₂₀ isa tyrosine residue, a glycine residue, a serine residue, or no residue,X₂₁ is a serine residue, an asparagine residue, a tryptophan residue, aglutamate residue, as aspartate residue, or no residue, X₂₂ is a serineresidue, an aspartate residue, a tryptophan residue, an alanine residue,an arginine residue, a threonine residue, a glutamine residue, a leucineresidue, a glutamate residue, or no residue, X₂₃ is a serine residue, aglycine residue, an asparagine residue, a threonine residue, atryptophan residue, a valine residue, an alanine residue, or anisoleucine residue, X₂₄ is an arginine residue, a glutamine residue, atyrosine residue, a valine residue, an alanine residue, a glycineresidue, a serine residue, a phenylalanine residue, or a tryptophanresidue, X₂₅ is an asparagine residue, a leucine residue, an aspartateresidue, a threonine residue, a tryptophan residue, a tyrosine residue,a valine residue, an alanine residue, or a histidine residue, X₂₆ is anaspartate residue, a serine residue, an asparagine residue, or aglutamine residue, X₂₇ is an alanine residue or a proline residue, X₂₈is an alanine residue or no residue, X₂₉ is a glutamate residue, atyrosine residue, a glycine residue, or no residue, X₃₀ is an arginineresidue, a serine residue, or no residue, X₃₁ is a glycine residue, anaspartate residue, a valine residue, a serine residue, or no residue,X₃₂ is a serine residue, an aspartate residue, a glycine residue, or noresidue, X₃₃ is a phenylalanine residue, an aspartate residue, atyrosine residue, a glycine residue, a serine residue, a histidineresidue, a tryptophan residue, or no residue, X₃₄ is a tryptophanresidue, an aspartate residue, a tyrosine residue, a serine residue, orno residue, X₃₅ is an aspartate residue, a glutamate residue, anarginine residue, a serine residue, a glycine residue, a tyrosineresidue, or a tryptophan residue, X₃₆ is a tyrosine residue, a lysineresidue, an isoleucine residue, a leucine residue or a phenylalanineresidue, X₃₇ is a tyrosine residue, a serine residue, a phenylalanineresidue, an aspartate residue, or a glycine residue, X₃₈ is a glycineresidue, an asparagine residue, or a tyrosine residue, X₃₉ is a valineresidue, a glycine residue, or a serine residue, and said antigenbinding protein binds specifically to human IGF-1R. In one embodiment,the isolated antigen binding protein comprises an amino acid sequenceselected from the group consisting of: a. a light chain CDR1 sequencethat differs by no more than a total of six amino acid additions,substitutions, and/or deletions from a CDR1 sequence of L1-L52 as shownin FIG. 4; b. a light chain CDR2 sequence that differs by no more than atotal of two amino acid additions, substitutions, and/or deletions froma CDR2 sequence of L1-L52 as shown in FIG. 5; c. a light chain CDR3sequence that differs by no more than a total of three amino acidadditions, substitutions, and/or deletions from a CDR3 sequence ofL1-L52 as shown in FIG. 6; d. a heavy chain CDR1 sequence that differsby no more than a total of two amino acid additions, substitutions,and/or deletions from a CDR1 sequence of H1-H52 as shown in FIG. 7; e. aheavy chain CDR2 sequence that differs by no more than a total of fiveamino acid additions, substitutions, and/or deletions from a CDR2sequence of H1-H52 as shown in FIG. 8; and f. a heavy chain CDR3sequence that differs by no more than a total of four amino acidadditions, substitutions, and/or deletions from a CDR3 sequence ofH1-H52 as shown in FIG. 9. In another embodiment, the isolated antigenbinding protein comprises an amino acid sequence selected from the groupconsisting of: a. a light chain CDR1 sequence that differs by no morethan a total of five amino acid additions, substitutions, and/ordeletions from a CDR1 sequence of L1-L52 as shown in FIG. 4; b. a lightchain CDR2 sequence that differs by no more than a total of one aminoacid addition, substitution, or deletion from a CDR2 sequence of L1-L52as shown in FIG. 5; c. a light chain CDR3 sequence that differs by nomore than a total of two amino acid additions, substitutions, and/ordeletions from a CDR3 sequence of L1-L52 as shown in FIG. 6; d. a heavychain CDR1 sequence that differs by no more than a total of one aminoacid addition, substitution, or deletion from a CDR1 sequence of H1-H52as shown in FIG. 7; e. a heavy chain CDR2 sequence that differs by nomore than a total of four amino acid additions, substitutions, and/ordeletions from a CDR2 sequence of H1-H52 as shown in FIG. 8; and f. aheavy chain CDR3 sequence that differs by no more than a total of threeamino acid additions, substitutions, and/or deletions from a CDR3sequence of H1-H52 as shown in FIG. 9. In another embodiment, theisolated antigen binding protein comprises an amino acid sequenceselected from the group consisting of: a. a light chain CDR1 sequencethat differs by no more than a total of four amino acid additions,substitutions, and/or deletions from a CDR1 sequence of L1-L52 as shownin FIG. 4; b. a light chain CDR2 sequence of L1-L52 as shown in FIG. 5;c. a light chain CDR3 sequence that differs by no more than a total ofone amino acid addition, substitution, or deletion from a CDR3 sequenceof L1-L52 as shown in FIG. 6; d. a heavy chain CDR1 sequence of H1-H52as shown in FIG. 7; e. a heavy chain CDR2 sequence that differs by nomore than a total of three amino acid additions, substitutions, and/ordeletions from a CDR2 sequence of H1-H52 as shown in FIG. 8; and f. aheavy chain CDR3 sequence that differs by no more than a total of twoamino acid additions, substitutions, and/or deletions from a CDR3sequence of H1-H52 as shown in FIG. 9. In another embodiment, theisolated antigen binding protein comprises an amino acid sequenceselected from the group consisting of: a. a light chain CDR1 sequencethat differs by no more than a total of three amino acid additions,substitutions, and/or deletions from a CDR1 sequence of L1-L52 as shownin FIG. 4; b. a light chain CDR3 sequence of L1-L52 as shown in FIG. 6;c. a heavy chain CDR2 sequence that differs by no more than a total oftwo amino acid additions, substitutions, and/or deletions from a CDR2sequence of H1-H52 as shown in FIG. 8; and d. a heavy chain CDR3sequence that differs by no more than a total of one amino acidaddition, substitution, or deletion from a CDR3 sequence of H1-H52 asshown in FIG. 9. In another embodiment, the isolated antigen bindingprotein comprises an amino acid sequence selected from the groupconsisting of: a. a light chain CDR1 sequence that differs by no morethan a total of two amino acid additions, substitutions, and/ordeletions from a CDR1 sequence of L1-L52 as shown in FIG. 4; b. a heavychain CDR2 sequence that differs by no more than a total of one aminoacid addition, substitution, or deletion from a CDR2 sequence of H1-H52as shown in FIG. 8; and c. a heavy chain CDR3 sequence of H1-H52 asshown in FIG. 9. In another embodiment, the isolated antigen bindingprotein comprises an amino acid sequence selected from the groupconsisting of: a. a light chain CDR1 sequence that differs by no morethan a total of one amino acid addition, substitution, or deletion froma CDR1 sequence of L1-L52 as shown in FIG. 4; and b. a heavy chain CDR2sequence of H1-H52 as shown in FIG. 8. In another embodiment, theisolated antigen binding protein comprises a CDR1 sequence of L1-L52 asshown in FIG. 4. In another embodiment, the isolated antigen bindingprotein comprises a sequence selected from the group consisting of: a. alight chain CDR1 sequence selected from the group consisting of: i.RSSQSLLHSNGYNYLD; ii. RASQ(G/S)(IN)(G/S)X(Y/F)L(A/N); and iii.RSSQS(L/I)XXXXX; b. a light chain CDR2 sequence selected from the groupconsisting of: i. LGSNRAS; ii. AASTLQS; and iii. EDNXRPS; c. a heavychain CDR1 sequence selected from the group consisting of: i. SSNWWS;ii. XYYWS; and iii. SYAM(S/H); and d. a heavy chain CDR2 sequenceselected from the group consisting of: i.(E/I)(I/V)(Y/N)(H/Y)SGST(N/Y)YNPSLKS; and ii. XIS(G/S)SG(G/S)STYYADSVKG;wherein amino acid residue symbols enclosed in parentheses identifyalternative residues for the same position in a sequence, each X isindependently any amino acid residue, and each Z is independently aglycine residue, an alanine residue, a valine residue, a leucineresidue, an isoleucine residue, a proline residue, a phenylalanineresidue, a methionine residue, a tryptophan residue, or a cysteineresidue. In another embodiment, the isolated antigen binding proteincomprises a heavy chain CDR3 sequence that differs by no more than atotal of two amino acid additions, substitutions, and/or deletions froma CDR3 sequence of H1-H52 as shown in FIG. 9. In another embodiment, theisolated antigen binding protein comprises a heavy chain CDR3 sequencethat differs by no more than a total of one amino acid addition,substitution, or deletion from a CDR3 sequence of H1-H52 as shown inFIG. 9. In another embodiment, the isolated antigen binding proteincomprises a heavy chain CDR3 sequence of H1-H52 as shown in FIG. 9. Inanother embodiment, the isolated antigen binding protein comprises twoamino acid sequences selected from the group consisting of: a. a lightchain CDR1 sequence that differs by no more than a total of six aminoacid additions, substitutions, and/or deletions from a CDR1 sequence ofL1-L52 as shown in FIG. 4; b. a light chain CDR2 sequence that differsby no more than a total of two amino acid additions, substitutions,and/or deletions from a CDR2 sequence of L1-L52 as shown in FIG. 5; c. alight chain CDR3 sequence that differs by no more than a total of threeamino acid additions, substitutions, and/or deletions from a CDR3sequence of L1-L52 as shown in FIG. 6; d. a heavy chain CDR1 sequencethat differs by no more than a total of two amino acid additions,substitutions, and/or deletions from a CDR1 sequence of H1-H52 as shownin FIG. 7; e. a heavy chain CDR2 sequence that differs by no more than atotal of five amino acid additions, substitutions, and/or deletions froma CDR2 sequence of H1-H52 as shown in FIG. 8; and f. a heavy chain CDR3sequence that differs by no more than a total of four amino acidadditions, substitutions, and/or deletions from a CDR3 sequence ofH1-H52 as shown in FIG. 9. In another embodiment, the isolated antigenbinding protein comprises three amino acid sequences selected from thegroup consisting of: a. a light chain CDR1 sequence that differs by nomore than a total of six amino acid additions, substitutions, and/ordeletions from a CDR1 sequence of L1-L52 as shown in FIG. 4; b. a lightchain CDR2 sequence that differs by no more than a total of two aminoacid additions, substitutions, and/or deletions from a CDR2 sequence ofL1-L52 as shown in FIG. 5; c. a light chain CDR3 sequence that differsby no more than a total of three amino acid additions, substitutions,and/or deletions from a CDR3 sequence of L1-L52 as shown in FIG. 6; d. aheavy chain CDR1 sequence that differs by no more than a total of twoamino acid additions, substitutions, and/or deletions from a CDR1sequence of H1-H52 as shown in FIG. 7; e. a heavy chain CDR2 sequencethat differs by no more than a total of five amino acid additions,substitutions, and/or deletions from a CDR2 sequence of H1-H52 as shownin FIG. 8; and f. a heavy chain CDR3 sequence that differs by no morethan a total of four amino acid additions, substitutions, and/ordeletions from a CDR3 sequence of H1-H52 as shown in FIG. 9. In anotherembodiment, the isolated antigen binding protein comprises four aminoacid sequences selected from the group consisting of: a. a light chainCDR1 sequence that differs by no more than a total of six amino acidadditions, substitutions, and/or deletions from a CDR1 sequence ofL1-L52 as shown in FIG. 4; b. a light chain CDR2 sequence that differsby no more than a total of two amino acid additions, substitutions,and/or deletions from a CDR2 sequence of L1-L52 as shown in FIG. 5; c. alight chain CDR3 sequence that differs by no more than a total of threeamino acid additions, substitutions, and/or deletions from a CDR3sequence of L1-L52 as shown in FIG. 6; d. a heavy chain CDR1 sequencethat differs by no more than a total of two amino acid additions,substitutions, and/or deletions from a CDR1 sequence of H1-H52 as shownin FIG. 7; e. a heavy chain CDR2 sequence that differs by no more than atotal of five amino acid additions, substitutions, and/or deletions froma CDR2 sequence of H1-H52 as shown in FIG. 8; and f. a heavy chain CDR3sequence that differs by no more than a total of four amino acidadditions, substitutions, and/or deletions from a CDR3 sequence ofH1-H52 as shown in FIG. 9. In another embodiment, the isolated antigenbinding protein comprises five amino acid sequences selected from thegroup consisting of: a. a light chain CDR1 sequence that differs by nomore than a total of six amino acid additions, substitutions, and/ordeletions from a CDR1 sequence of L1-L52 as shown in FIG. 4; b. a lightchain CDR2 sequence that differs by no more than a total of two aminoacid additions, substitutions, and/or deletions from a CDR2 sequence ofL1-L52 as shown in FIG. 5; c. a light chain CDR3 sequence that differsby no more than a total of three amino acid additions, substitutions,and/or deletions from a CDR3 sequence of L1-L52 as shown in FIG. 6; d. aheavy chain CDR1 sequence that differs by no more than a total of twoamino acid additions, substitutions, and/or deletions from a CDR1sequence of H1-H52 as shown in FIG. 7; e. a heavy chain CDR2 sequencethat differs by no more than a total of five amino acid additions,substitutions, and/or deletions from a CDR2 sequence of H1-H52 as shownin FIG. 8; and f. a heavy chain CDR3 sequence that differs by no morethan a total of four amino acid additions, substitutions, and/ordeletions from a CDR3 sequence of H1-H52 as shown in FIG. 9. In anotherembodiment, the isolated antigen binding protein comprises: a. a lightchain CDR1 sequence that differs by no more than a total of six aminoacid additions, substitutions, and/or deletions from a CDR1 sequence ofL1-L52 as shown in FIG. 4; b. a light chain CDR2 sequence that differsby no more than a total of two amino acid additions, substitutions,and/or deletions from a CDR2 sequence of L1-L52 as shown in FIG. 5; c. alight chain CDR3 sequence that differs by no more than a total of threeamino acid additions, substitutions, and/or deletions from a CDR3sequence of L1-L52 as shown in FIG. 6; d. a heavy chain CDR1 sequencethat differs by no more than a total of two amino acid additions,substitutions, and/or deletions from a CDR1 sequence of H1-H52 as shownin FIG. 7; e. a heavy chain CDR2 sequence that differs by no more than atotal of five amino acid additions, substitutions, and/or deletions froma CDR2 sequence of H1-H52 as shown in FIG. 8; and f. a heavy chain CDR3sequence that differs by no more than a total of four amino acidadditions, substitutions, and/or deletions from a CDR3 sequence ofH1-H52 as shown in FIG. 9. In another embodiment, the isolated antigenbinding protein comprises either: a. a light chain variable domaincomprising: i. a light chain CDR1 sequence shown in FIG. 4; ii. a lightchain CDR2 sequence shown in FIG. 5; and iii. a light chain CDR3sequence shown in FIG. 6; b. a heavy chain variable domain comprising:i. a heavy chain CDR1 sequence shown in FIG. 7; ii. a heavy chain CDR2sequence shown in FIG. 8; and iii. a heavy chain CDR3 sequence shown inFIG. 9; or c. the light chain variable domain of (a) and the heavy chainvariable domain of (b). In another embodiment, the isolated antigenbinding protein comprises either: a. light chain CDR1, CDR2, and CDR3sequences that each is identical to the CDR1, CDR2, and CDR3 sequences,respectively, of the same light chain variable domain sequence selectedfrom the group consisting of L1-L52; b. heavy chain CDR1, CDR2, and CDR3sequences that each is identical to the CDR1, CDR2, and CDR3 sequences,respectively, of the same heavy chain variable domain sequence selectedfrom the group consisting of H1-H52; or c. the light chain CDR1, CDR2,and CDR3 sequences of (a) and the heavy chain CDR1, CDR2, and CDR3sequences of (b).

In another aspect, the present invention provides an isolated antigenbinding protein comprising either: a. a light chain variable domainsequence selected from the group consisting of: i. a sequence of aminoacids at least 80% identical to a light chain variable domain sequenceof L1-L52 as shown in FIG. 2; ii. a sequence of amino acids comprisingat least 15 contiguous amino acid residues of a light chain variabledomain sequence of L1-L52 as shown in FIG. 2; iii. a sequence of aminoacids encoded by a polynucleotide sequence that is at least 80%identical to a polynucleotide sequence encoding a light chain variabledomain sequence of L1-L52 as shown in FIG. 1; and iv. a sequence ofamino acids encoded by a polynucleotide sequence that hybridizes undermoderately stringent conditions to the complement of a polynucleotideconsisting of a light chain variable domain sequence of L1-L52 as shownin FIG. 1; b. a heavy chain variable domain sequence selected from thegroup consisting of: i. a sequence of amino acids at least 80% identicalto a heavy chain variable domain sequence of H1-H52 as shown in FIG. 2;ii. a sequence of amino acids comprising at least 15 contiguous aminoacid residues of a heavy chain variable domain sequence of H1-H52 asshown in FIG. 2; iii. a sequence of amino acids encoded by apolynucleotide sequence that is at least 80% identical to apolynucleotide sequence encoding a heavy chain variable domain sequenceof H1-H52 as shown in FIG. 1; and iv. a sequence of amino acids encodedby a polynucleotide sequence that hybridizes under moderately stringentconditions to the complement of a polynucleotide consisting of a heavychain variable domain sequence of H1-H52 as shown in FIG. 1; or c. thelight chain variable domain of (a) and the heavy chain variable domainof (b); wherein said antigen binding protein binds to human IGF-1R. Inone embodiment, the isolated antigen binding protein comprises either:a. a light chain variable domain sequence selected from the groupconsisting of: i. a sequence of amino acids at least 85% identical to alight chain variable domain sequence of L1-L52 as shown in FIG. 2; ii. asequence of amino acids comprising at least 25 contiguous amino acidresidues of a light chain variable domain sequence of L1-L52 as shown inFIG. 2; iii. a sequence of amino acids encoded by a polynucleotidesequence that is at least 85% identical to a polynucleotide sequenceencoding a light chain variable domain sequence of L1-L52 as shown inFIG. 1; and iv. a sequence of amino acids encoded by a polynucleotidesequence that hybridizes under highly stringent conditions to thecomplement of a polynucleotide consisting of a light chain variabledomain sequence of L1-L52 as shown in FIG. 1; b. a heavy chain variabledomain sequence selected from the group consisting of: i. a sequence ofamino acids at least 85% identical to a heavy chain variable domainsequence of H1-H52 as shown in FIG. 2; ii. a sequence of amino acidscomprising at least 25 contiguous amino acid residues of a heavy chainvariable domain sequence of H1-H52 as shown in FIG. 2; iii. a sequenceof amino acids encoded by a polynucleotide sequence that is at least 85%identical to a polynucleotide sequence encoding a heavy chain variabledomain sequence of H1-H52 as shown in FIG. 1; and iv. a sequence ofamino acids encoded by a polynucleotide sequence that hybridizes underhighly stringent conditions to the complement of a polynucleotideconsisting of a heavy chain variable domain sequence of H1-H52 as shownin FIG. 1; or c) the light chain variable domain of (a) and the heavychain variable domain of (b). In another embodiment, the isolatedantigen binding protein comprises either: a. a light chain variabledomain sequence selected from the group consisting of: i. a sequence ofamino acids at least 90% identical to a light chain variable domainsequence of L1-L52 as shown in FIG. 2; ii. a sequence of amino acidscomprising at least 35 contiguous amino acid residues of a light chainvariable domain sequence of L1-L52 as shown in FIG. 2; and iii. asequence of amino acids encoded by a polynucleotide sequence that is atleast 90% identical to a polynucleotide sequence encoding a light chainvariable domain sequence of L1-L52 as shown in FIG. 1; and b. a heavychain variable domain sequence selected from the group consisting of: i.a sequence of amino acids at least 90% identical to a heavy chainvariable domain sequence of H1-H52 as shown in FIG. 2; ii. a sequence ofamino acids comprising at least 35 contiguous amino acid residues of aheavy chain variable domain sequence of H1-H52 as shown in FIG. 2; andiii. a sequence of amino acids encoded by a polynucleotide sequence thatis at least 90% identical to a polynucleotide sequence encoding a heavychain variable domain sequence of H1-H52 as shown in FIG. 1; or c) thelight chain variable domain of (a) and the heavy chain variable domainof (b). In another embodiment, the isolated antigen binding proteincomprises either: a. a light chain variable domain sequence selectedfrom the group consisting of: i. a sequence of amino acids at least 95%identical to a light chain variable domain sequence of L1-L52 as shownin FIG. 2; ii. a sequence of amino acids comprising at least 50contiguous amino acid residues of a light chain variable domain sequenceof L1-L52 as shown in FIG. 2; and iii. a sequence of amino acids encodedby a polynucleotide sequence that is at least 95% identical to apolynucleotide sequence encoding a light chain variable domain sequenceof L1-L52 as shown in FIG. 1; and b. a heavy chain variable domainsequence selected from the group consisting of: i. a sequence of aminoacids at least 95% identical to a heavy chain variable domain sequenceof H1-H52 as shown in FIG. 2; ii. a sequence of amino acids comprisingat least 50 contiguous amino acid residues of a heavy chain variabledomain sequence of H1-H52 as shown in FIG. 2; and iii. a sequence ofamino acids encoded by a polynucleotide sequence that is at least 95%identical to a polynucleotide sequence encoding a heavy chain variabledomain sequence of H1-H52 as shown in FIG. 1; or c) the light chainvariable domain of (a) and the heavy chain variable domain of (b). Inanother embodiment, the isolated antigen binding protein compriseseither: a. a light chain variable domain sequence selected from thegroup consisting of: i. a sequence of amino acids at least 97% identicalto a light chain variable domain sequence of L1-L52 as shown in FIG. 2;ii. a sequence of amino acids comprising at least 75 contiguous aminoacid residues of a light chain variable domain sequence of L1-L52 asshown in FIG. 2; and iii. a sequence of amino acids encoded by apolynucleotide sequence that is at least 97% identical to apolynucleotide sequence encoding a light chain variable domain sequenceof L1-L52 as shown in FIG. 1; and b. a heavy chain variable domainsequence selected from the group consisting of: i. a sequence of aminoacids at least 97% identical to a heavy chain variable domain sequenceof H1-H52 as shown in FIG. 2; ii. a sequence of amino acids comprisingat least 75 contiguous amino acid residues of a heavy chain variabledomain sequence of H1-H52 as shown in FIG. 2; and iii. a sequence ofamino acids encoded by a polynucleotide sequence that is at least 97%identical to a polynucleotide sequence encoding a heavy chain variabledomain sequence of H1-H52 as shown in FIG. 1; or c) the light chainvariable domain of (a) and the heavy chain variable domain of (b). Inanother embodiment, the isolated antigen binding protein compriseseither: a. a light chain variable domain sequence selected from thegroup consisting of: i. a sequence of amino acids at least 99% identicalto a light chain variable domain sequence of L1-L52 as shown in FIG. 2;ii. a sequence of amino acids comprising at least 90 contiguous aminoacid residues of a light chain variable domain sequence of L1-L52 asshown in FIG. 2; and iii. a sequence of amino acids encoded by apolynucleotide sequence that is at least 99% identical to apolynucleotide sequence encoding a light chain variable domain sequenceof L1-L52 as shown in FIG. 1; and b. a heavy chain variable domainsequence selected from the group consisting of: i. a sequence of aminoacids at least 99% identical to a heavy chain variable domain sequenceof H1-H52 as shown in FIG. 2; ii. a sequence of amino acids comprisingat least 90 contiguous amino acid residues of a heavy chain variabledomain sequence of H1-H52 as shown in FIG. 2; and iii. a sequence ofamino acids encoded by a polynucleotide sequence that is at least 99%identical to a polynucleotide sequence encoding a heavy chain variabledomain sequence of H1-H52 as shown in FIG. 1; or c. the light chainvariable domain of (a) and the heavy chain variable domain of (b). Inanother embodiment, the isolated antigen binding protein compriseseither: a. a light chain variable domain sequence selected from thegroup consisting of L1-L52 as shown in FIG. 2; b. a heavy chain variabledomain sequence selected from the group consisting of H1-H52 as shown inFIG. 3; or c. the light chain variable domain of (a) and the heavy chainvariable domain of (b). In another embodiment, the isolated antigenbinding protein comprises a combination of a light chain variable domainand a heavy chain variable domain selected from the group ofcombinations consisting of: L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7,L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16,L17H17, L18H18, L19H19, L20, H20, L21H21, L22H22, L23H23, L24H24,L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31, L32H32, L33H33,L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42,L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51,and L52H52. In another embodiment, the isolated antigen binding proteinfurther comprises: a. the kappa light chain constant sequence of FIG.13, b. the IgG1 heavy chain constant sequence of FIG. 13, or c. thekappa light chain constant sequence of FIG. 13 and the IgG1 heavy chainconstant sequence of FIG. 13. In another embodiment, the isolatedantigen binding protein, when bound to IGF-1R: a. inhibits IGF-1R; b.activates IGF-1R; c. cross-competes with a reference antibody forbinding to IGF-1R; d. binds to the same epitope of IGF-1R as saidreference antibody; e. binds to IGF-1R with substantially the same Kd assaid reference antibody; or f. binds to IGF-1R with substantially thesame off rate as said reference antibody; wherein said referenceantibody comprises a combination of light chain and heavy chain variabledomain sequences selected from the group of combinations consisting ofL1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11,L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20,H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28,L29H29, L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37,L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46,L47H47, L48H48, L49H49, L50H50, L51H51, and L52H52. In anotherembodiment, the isolated antigen binding protein, when bound to a humanIGF-1R, inhibits binding of IGF-1 and/or IGF-2 to said human IGF-1R. Inanother embodiment, the isolated antigen binding protein inhibits thegrowth of a cancer cell by greater than about 80% in the presence of agrowth stimulant selected from the group consisting of serum, IGF-1, andIGF-2. In another embodiment, said cancer cell is an MCF-7 human breastcancer cell. In another embodiment, the isolated antigen binding proteinbinds to human IGF-1R with a selectivity that is at least fifty timesgreater than its selectivity for human insulin receptor. In anotherembodiment, the isolated antigen binding protein inhibits tumor growthin vivo. In another embodiment, the isolated antigen binding proteininhibits IGF-1R mediated tyrosine phosphorylation. In anotherembodiment, the isolated antigen binding protein specifically binds tothe IGF-1R of a non-human primate, a cynomologous monkey, a chimpanzee,a non-primate mammal, a rodent, a mouse, a rat, a hamster, a guinea pig,a cat, or a dog. In another embodiment, the isolated antigen bindingprotein comprises: a. a human antibody; b. a humanized antibody; c. achimeric antibody; d. a monoclonal antibody; e. a polyclonal antibody;f. a recombinant antibody; g. an antigen-binding antibody fragment; h. asingle chain antibody; i. a diabody; j. a triabody; k. a tetrabody; l. aFab fragment; m. a F(ab′)₂ fragment; n. a domain antibody; o. an IgDantibody; p. an IgE antibody; q. an IgM antibody; r. an IgG1 antibody;s. an IgG2 antibody; t. an IgG3 antibody; u. an IgG4 antibody; or v. anIgG4 antibody having at least one mutation in a hinge region thatalleviates a tendency to form intra-H chain disulfide bond.

In another aspect, the present invention provides an isolatedpolynucleotide comprising a sequence that encodes the light chain, theheavy chain, or both of said antigen binding protein. In one embodiment,said polynucleotide comprises a light chain variable domain nucleic acidsequence of FIG. 1 and/or a heavy chain variable domain nucleic acidsequence of FIG. 1. In another embodiment, a plasmid comprises saidisolated polynucleotide. In another embodiment, said plasmid is anexpression vector. In another embodiment, an isolated cell comprisessaid polynucleotide. In another embodiment, a chromosome of said cellcomprises said polynucleotide. In another embodiment, said cell is ahybridoma. In another embodiment, an expression vector comprises saidpolynucleotide. In another embodiment, said cell is a CHO cell. Inanother embodiment, the present invention provides a method of making anantigen binding protein that binds human IGF-1R, comprising incubatingsaid isolated cell under conditions that allow it to express saidantigen binding protein.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising the antigen binding protein. In one embodiment,the present invention provides a method of treating a condition in asubject comprising administering to said subject said pharmaceuticalcomposition, wherein said condition is treatable by reducing theactivity of IGF-1R in said subject. In another embodiment, said subjectis a human being. In another embodiment, said condition is multiplemyeloma, a liquid tumor, liver cancer, a thymus disorder, a T-cellmediated autoimmune disease, an endocronological disorder, ischemia, ora neurodegenerative disorder. In another embodidment, said liquid tumoris selected from the group consisting of acute lymphocytic leukemia(ALL) and chronic myelogenous leukemia (CML); wherein said liver canceris selected from the group consisting of hepatoma, hepatocellularcarcinoma, cholangiocarcinoma, angiosarcomas, hemangiosarcomas,hepatoblastoma; wherein said thymus disorder is selected from the groupconsisting of thymoma and thyroiditis, wherein said T-cell mediatedautoimmune disease is selected from the group consisting of MultipleSclerosis, Rheumatoid Arthritis, Systemic Lupus Erythematosus (SLE),Grave's Disease, Hashimoto's Thyroiditis, Myasthenia Gravis, Auto-ImmuneThyroiditis, Bechet's Disease, wherein said endocrinological disorder isselected from the group consisting of Type II Diabetes, hyperthyroidism,hypothyroidism, thyroiditis, hyperadrenocorticism, andhypoadrenocorticism; wherein said ischemia is post cardiac infarctischemia, or wherein said neurodegenerative disorder is Alzheimer'sDisease. In another embodiment, said condition is selected from thegroup consisting of acromegaly, bladder cancer, Wilm's tumor, ovariancancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer,prostate cancer, bone cancer, lung cancer, colorectal cancer, cervicalcancer, synovial sarcoma, diarrhea associated with metastatic carcinoid,vasoactive intestinal peptide secreting tumors, gigantism, psoriasis,atherosclerosis, smooth muscle restenosis of blood vessels,inappropriate microvascular proliferation, glioblastoma,medulloblastoma, head and neck squamous cell cancer, oral cancer, oralleukoplakia, prostate intraepithelial neoplasia, anal cancer, esophagealcancer, gastric cancer, bone cancer, metastatic cancer, polycythemiarubra vera, a benign condition related to oxidative stress, retinopathyof prematurity, Acute Respiratory Distress Syndrome, an overdose ofacetaminophen, bronchopulmonary dysplasia, cystic fibrosis, lungfibrosis, and diabetic retinopathy. In another embodiment, the methodfurther comprising administering to said subject a second treatment. Inanother embodiment, said second treatment is administered to saidsubject before and/or simultaneously with and/or after saidpharmaceutical composition is administered to said subject. In anotherembodiment, said second treatment comprises radiation treatment,surgery, or a second pharmaceutical composition. In another embodiment,said second pharmaceutical composition comprises an agent selected fromthe group consisting of a corticosteroid, an anti-emetic, ondansetronhydrochloride, granisetron hydrochloride, metroclopramide, domperidone,haloperidol, cyclizine, lorazepam, prochlorperazine, dexamethasone,levomepromazine, tropisetron, a cancer vaccine, a GM-CSF inhibitingagent, a GM-CSF DNA vaccine, a cell-based vaccine, a dendritic cellvaccine, a recombinant viral vaccine, a heat shock protein (HSP)vaccine, an allogeneic tumor vaccine, an autologous tumor vaccine, ananalgesic, ibuprofen, naproxen, choline magnesium trisalicylate, anoxycodone hydrochloride, an anti-angiogenic agent, an anti-vascularagent, bevacizumab, an anti-VEGF antibody, an anti-VEGF receptorantibody, a soluble VEGF receptor fragment, an anti-TWEAK antibody, ananti-TWEAK receptor antibody, a soluble TWEAK receptor fragment, AMG706, AMG 386, an anti-proliferative agent, a farnesyl proteintransferase inhibitor, an αvβ3 inhibitor, an αvβ5 inhibitor, a p53inhibitor, a Kit receptor inhibitor, a ret receptor inhibitor, a PDGFRinhibitor, a growth hormone secretion inhibitor, an angiopoietininhibitor, a tumor infiltrating macrophage-inhibiting agent, a c-fmsinhibiting agent, an anti-c-fms antibody, an CSF-1 inhibiting agent, ananti-CSF-1 antibody, a soluble c-fms fragment, pegvisomant, gemcitabine,panitumumab, irinothecan, and SN-38. In another embodiment, said methodcomprises administering to said subject a third treatment. In anotherembodiment, said condition is a cancer, said second treatment comprisesadministering panitumumab, and said third treatment comprisesadministering gemcitabine. In another embodiment, said condition isselected from the group consisting of acromegaly, bladder cancer, Wilm'stumor, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia,breast cancer, prostate cancer, bone cancer, lung cancer, colorectalcancer, cervical cancer, synovial sarcoma, diarrhea associated withmetastatic carcinoid, vasoactive intestinal peptide secreting tumors,gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of bloodvessels, inappropriate microvascular proliferation, glioblastoma,medulloblastoma, head and neck squamous cell cancer, oral cancer, oralleukoplakia, prostate intraepithelial neoplasia, anal cancer, esophagealcancer, gastric cancer, bone cancer, metastatic cancer, polycythemiarubra vera, a benign condition related to oxidative stress, retinopathyof prematurity, Acute Respiratory Distress Syndrome, an overdose ofacetaminophen, bronchopulmonary dysplasia, cystic fibrosis, lungfibrosis, and diabetic retinopathy.

In another aspect, the present invention provides a method of increasingthe longevity of a subject comprising administering to said subject saidpharmaceutical composition.

In another aspect, the present invention provides a method of decreasingIGF-1R activity in a subject in need thereof comprising administering tosaid subject said pharmaceutical composition.

In another aspect, the present invention provides a method of decreasingIGF-1R signaling in a subject in need thereof comprising administeringto said subject said pharmaceutical composition.

In another aspect, the present invention provides a method of inhibitingthe binding of IGF-1 and/or IGF-2 to IGF-1R in a subject in need thereofcomprising administering to said subject said pharmaceuticalcomposition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions, kits, and methods relatingto molecules that bind to the Insulin-Like Growth Factor Receptor(“IGF-1R”), including molecules that agonize or antagonize IGF-1R, suchas anti-IGF-1R antibodies, antibody fragments, and antibody derivatives,e.g., antagonistic anti-IGF-1R antibodies, antibody fragments, orantibody derivatives. Also provided are nucleic acids, and derivativesand fragments thereof, comprising a sequence of nucleotides that encodesall or a portion of a polypeptide that binds to IGF-1R, e.g., a nucleicacid encoding all or part of an anti-IGF-1R antibody, antibody fragment,or antibody derivative, plasmids and vectors comprising such nucleicacids, and cells or cell lines comprising such nucleic acids and/orvectors and plasmids. The provided methods include, for example, methodsof making, identifying, or isolating molecules that bind to IGF-1R, suchas anti-IGF-1R antibodies, methods of determining whether a moleculebinds to IGF-1R, methods of determining whether a molecule agonizes orantagonizes IGF-1R, methods of making compositions, such aspharmaceutical compositions, comprising a molecule that binds to IGF-1R,and methods for administering a molecule that binds IGF-1R to a subject,for example, methods for treating a condition mediated by IGF-1R, andfor agonizing or antagonizing a biological activity of IGF-1R, IGF-1,and/or IGF-2 in vivo or in vitro.

Polynucleotide and polypeptide sequences are indicated using standardone- or three-letter abbreviations. Unless otherwise indicated,polypeptide sequences have their amino termini at the left and theircarboxy termini at the right and single-stranded nucleic acid sequences,and the top strand of double-stranded nucleic acid sequences, have their5′ termini at the left and their 3′ termini at the right. A particularpolypeptide or polynucleotide sequence also can be described byexplaining how it differs from a reference sequence.

Polynucleotide and polypeptide sequences of particular light and heavychain variable domains are shown in FIGS. 1, 2 and 3, where they arelabeled, for example, L1 (“light chain variable domain 1”), H1 (“heavychain variable domain 1”), etc. Antibodies comprising a light chain andheavy chain from FIGS. 2 and 3 are indicated by combining the name ofthe light chain and the name of the heavy chain variable domains. Forexample, “L4H7,” indicates an antibody comprising the light chainvariable domain of L4 and the heavy chain variable domain of H7.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art. The methodsand techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The terminology used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques can be used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings.

The term “isolated molecule” (where the molecule is, for example, apolypeptide, a polynucleotide, or an antibody) is a molecule that byvirtue of its origin or source of derivation (1) is not associated withnaturally associated components that accompany it in its native state,(2) is substantially free of other molecules from the same species (3)is expressed by a cell from a different species, or (4) does not occurin nature. Thus, a molecule that is chemically synthesized, orsynthesized in a cellular system different from the cell from which itnaturally originates, will be “isolated” from its naturally associatedcomponents. A molecule also may be rendered substantially free ofnaturally associated components by isolation, using purificationtechniques well known in the art. Molecule purity or homogeneity may beassayed by a number of means well known in the art. For example, thepurity of a polypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

The terms “IGF-1R inhibitor” and “IGF-1R antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of IGF-1R. Conversely, an “IGF-1R agonist” is a moleculethat detectably increases at least one function of IGF-1R. Theinhibition caused by an IGF-1R inhibitor need not be complete so long asit is detectable using an assay. Any assay of a function of IGF-1R canbe used, examples of which are provided herein. Examples of functions ofIGF-1R that can be inhibited by an IGF-1R inhibitor, or increased by anIGF-1R agonist, include binding to IGF-1, IGF-12, and/or anotherIGF-1R-activating molecule, kinase activity, downstream signaling, andso on. Examples of types of IGF-1R inhibitors and IGF-1R agonistsinclude, but are not limited to, IGF-1R binding polypeptides such asantigen binding proteins (e.g., IGF-1R inhibiting antigen bindingproteins), antibodies, antibody fragments, and antibody derivatives.

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion as comparedto a corresponding full-length protein. Fragments can be, for example,at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100,150 or 200 amino acids in length. Fragments can also be, for example, atmost 1,000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50,40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids in length. A fragmentcan further comprise, at either or both of its ends, one or moreadditional amino acids, for example, a sequence of amino acids from adifferent naturally-occurring protein (e.g., an Fc or leucine zipperdomain) or an artificial amino acid sequence (e.g., an artificial linkersequence).

Polypeptides of the invention include polypeptides that have beenmodified in any way and for any reason, for example, to: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties. Analogs include muteins of a polypeptide. Forexample, single or multiple amino acid substitutions (e.g., conservativeamino acid substitutions) may be made in the naturally occurringsequence (e.g., in the portion of the polypeptide outside the domain(s)forming intermolecular contacts. A “conservative amino acidsubstitution” is one that does not substantially change the structuralcharacteristics of the parent sequence (e.g., a replacement amino acidshould not tend to break a helix that occurs in the parent sequence, ordisrupt other types of secondary structure that characterize the parentsequence or are necessary for its functionality). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed., W. H. Freeman and Company, New York (1984)); Introduction toProtein Structure (C. Branden and J. Tooze, eds., Garland Publishing,New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991), whichare each incorporated herein by reference.

The present invention also provides non-peptide analogs of IGF-1Rbinding polypeptides. Non-peptide analogs are commonly used in thepharmaceutical industry as drugs with properties analogous to those ofthe template peptide. These types of non-peptide compound are termed“peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res.15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al.J. Med. Chem. 30:1229 (1987), which are incorporated herein byreference. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a desired biochemical property or pharmacological activity), such asa human antibody, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and—CH₂SO—, by methods well known in the art. Systematic substitution ofone or more amino acids of a consensus sequence with a D-amino acid ofthe same type (e.g., D-lysine in place of L-lysine) may also be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference), for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

A “variant” of a polypeptide (e.g., an antibody) comprises an amino acidsequence wherein one or more amino acid residues are inserted into,deleted from and/or substituted into the amino acid sequence relative toanother polypeptide sequence. Variants of the invention include fusionproteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety such as, for example, polyethylene glycol, albumin(e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

Naturally occurring immunoglobulin chains exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. From N-terminus to C-terminus, both light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat et al. in Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed., US Dept. of Health and Human Services, PHS, NIH,NIH Publication no. 91-3242, 1991.

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and complementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies, triabodies,tetrabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H)1 domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H)1 domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634, 6,696,245, US App. Pub.No. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward etal., Nature 341:544-546, 1989).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci.USA 85:5879-83). Diabodies are bivalent antibodies comprising twopolypeptide chains, wherein each polypeptide chain comprises V_(H) andV_(L) domains joined by a linker that is too short to allow for pairingbetween two domains on the same chain, thus allowing each domain to pairwith a complementary domain on another polypeptide chain (see, e.g.,Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljaket al., 1994, Structure 2:1121-23). If the two polypeptide chains of adiabody are identical, then a diabody resulting from their pairing willhave two identical antigen binding sites. Polypeptide chains havingdifferent sequences can be used to make a diabody with two differentantigen binding sites. Similarly, tribodies and tetrabodies areantibodies comprising three and four polypeptide chains, respectively,and forming three and four antigen binding sites, respectively, whichcan be the same or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. in Sequences of Proteins of Immunological Interest, 5th Ed., USDept. of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, 1991. One or more CDRs may be incorporated into a moleculeeither covalently or noncovalently to make it an antigen bindingprotein. An antigen binding protein may incorporate the CDR(s) as partof a larger polypeptide chain, may covalently link the CDR(s) to anotherpolypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRspermit the antigen binding protein to specifically bind to a particularantigen of interest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-IGF-1R antibody. In another embodiment, all of theCDRs are derived from a human anti-IGF-1R antibody. In anotherembodiment, the CDRs from more than one human anti-IGF-1R antibodies aremixed and matched in a chimeric antibody. For instance, a chimericantibody may comprise a CDR1 from the light chain of a first humananti-IGF-1R antibody, a CDR2 and a CDR3 from the light chain of a secondhuman anti-IGF-1R antibody, and the CDRs from the heavy chain from athird anti-IGF-1R antibody. Further, the framework regions may bederived from one of the same anti-IGF-1R antibodies, from one or moredifferent antibodies, such as a human antibody, or from a humanizedantibody. In one example of a chimeric antibody, a portion of the heavyand/or light chain is identical with, homologous to, or derived from anantibody from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is/are identicalwith, homologous to, or derived from an antibody(-ies) from anotherspecies or belonging to another antibody class or subclass. Alsoincluded are fragments of such antibodies that exhibit the desiredbiological activity (i.e., the ability to specifically bind IGF-1R).See, e.g., U.S. Pat. No. 4,816,567 and Morrison, 1985, Science229:1202-07.

A “neutralizing antibody” or “an inhibitory antibody” is an antibodythat inhibits the binding of IGF-1R to IGF-I and/or IGF-2 when an excessof the anti-IGF-1R antibody reduces the amount of IGF-I and/or IGF-2bound to IGF-1R by at least about 20% using the assay described inExample 9. In various embodiments, the antibody reduces the amount ofIGF-I and/or IGF-2 bound to IGF-1R by at least 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

An “activating antibody” is an antibody that activates IGF-1R by atleast about 20% when added to a cell, tissue or organism expressingIGF-1R, where “100% activation” is the level of activation achievedunder physiological conditions by the same molar amount of IGF-1 and/orIGF-2. In various embodiments, the antibody activates IGF-1R activity byat least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%,200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, or 1000%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques well-known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Methods to identify protein sequences that fold into aknown three-dimensional structure are known. See, e.g., Bowie et al.,1991, Science 253:164.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human IGF-1R) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof, of the invention.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientiation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293, 293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a hostcell is a cultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but to theprogeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

IGF-1R

IGF-1R is a transmembrane receptor tyrosine kinase (Blume-Jensen et al.,2001, Nature 411:355-65). The human IGF-1R is synthesized as a 1367amino acid precursor polypeptide that includes a 30 amino acid signalpeptide removed during translocation into the endoplasmic reticulum(Swiss-Prot: P08069). The IGF-1R proreceptor is glycosylated and cleavedby a protease at positions 708-711 (counting from the first amino acidfollowing the signal peptide sequence) during maturation in the ER-golgiresulting in the formation of an α-chain (1-707) and a β-chain(712-1337) that remain linked by disulfide bonds (Bhaumick et al., 1981,Proc Natl Acad Sci USA 78:4279-83, Chernausek et al., 1981, Biochemistry20:7345-50, Jacobs et al., 1983, Proc Natl Acad Sci USA 80:1228-31,LeBon et al., 1986, J Biol Chem 261:7685-89, Elleman, et al., 2000,Biochem J 347:771-79). The predominant form of the IGF-1R (and INSR)that exists on the cell-surface is a proteolytically processed andglycosylated (αβ)₂ dimer joined covalently by one or more disulfidebonds.

The extracellular portion of the IGF-1R consists of the α-chain and 191amino acids of the β-chain (712-905). The receptor contains a singletransmembrane spanning sequence (906-929) and a 408-residue cytoplasmicdomain that includes a functional tyrosine kinase (Rubin et al., 1983,Nature 305:438-440). Comparative sequence analysis has revealed that theIGF-1R is composed of 11 distinct structural motifs (reviewed by Adamset al., 2000, Cell Mol Life Sci 57:1050-93, Marino-Buslje et al., 1998,FEBS Ltrs 441:331-36, Ward et al., 2001, BMC Bioinformatics 2:4). TheN-terminal half of the extracellular domain contains two homologousdomains referred to as L1 (1-151) and L2 (299-461) (Ward et al., 2001,supra) separated by a cysteine-rich (CR) region (152-298) consisting ofseveral structural modules with disulfide linkages that align withrepeating units present in the TNF receptor and laminin (Ward et al.,1995, Proteins 22:141-53). The crystal structure of the L1-CR-L2 domainhas been solved (Garrett et al., 1998, Nature 394:395-99). The L2 domainis followed by three fibronectin type III domains (Marino-Buslje et al.,1998, supra, Mulhern et al., 1998, Trends Biochem Sci 23:465-66, Ward etal., 1999, Growth Factors 16:315-22). The first FnIII domain (FnIII-1,461-579) is 118 amino acids in length. The second FnIII domain (FnIII-2,580-798) is disrupted by a major insert sequence (ID) of about 120 aminoacids in length. The ID domain includes a furin protease cleavage sitethat separates the α and β chains of the mature receptor. The thirdFnIII domain (FnIII-3) is located entirely in the β-chain (799-901)terminating several residues before the transmembrane sequence. Thecatalytic domain of the IGF-1R tyrosine kinase is located between aminoacids positions 973-1229, and its structure has been solved (Favelyukiset al., 2001, Nature Structural Biol 8:1058-63, Pautsch et al., 2001,Structure 9:955-65). The kinase is flanked by two regulatory regions,the juxtamembrane region (930-972) and a 108 amino acid C-terminal tail(1220-1337) (Surmacz et al., 1995, Experimental Cell Res 218:370-80,Hongo et al., 1996, Oncogene 12:1231-38). The two regulatory regionscontain tyrosine residues that serve as docking sites for signaltransducing proteins when phosphorylated by the activated IGF-1Rtyrosine kinase (reviewed by Baserga (ed.), 1998 The IGF-1 Receptor inNormal and Abnormal Growth, Hormones and Growth Factors in Developmentand Neoplasia, Wiley-Liss, Inc., Adams et al., 2000, Cell Mol Life Sci57:1050-93).

The IGF-1R amino acid sequence is about 70% identical to the insulinreceptor (INSR; Swiss-Prot: P06213). The highest homology between thereceptors is located in the tyrosine kinase domain (84%); the lowestidentity is in the CR region and the C-terminus. The IGF-1R is alsohighly related (˜55% identical) to the insulin related receptor (IRR;Swiss-Prot: P14616).

Human IGF-1R can be activated by the insulin-like growth factors, IGF-1and IGF-2 and insulin (INS) (Hill et al., 1985, Pediatric Research19:879-86). IGF-1 and IGF-2 are encoded nonallelic genes (Brissenden etal., 1984, Nature 310: 781-8, Bell et al., 1985, Proceedings of theNational Academy of Sciences of the United States of America 82:6450-54), and both genes express alternative proteins related bydifferential RNA splicing and protein processing. The most common andwell-studied mature forms of IGF-1 and IGF-2 are respectively 70 and 67amino acids in length (Jansen et al., 1983, Nature 306:609-11, Dull etal., 1984, Nature 310: 777-81). These proteins (and their isoforms) areidentical at 11/21 positions to the insulin A-peptide, and identical at12/30 positions with the insulin B-peptide.

IGF-1R is expressed in all cells types in the normal adult animal exceptfor liver hepatocytes and mature B-cells. Human blood plasma containshigh concentrations of IGF-1 and IGF-2, and IGF-1 can be detected inmost tissues. The receptor is an integral component of the physiologicalmechanism controlling organ size and homeostasis. Without being bound toa particular theory, the “Somatomedin Hypothesis” states that GrowthHormone (GH) mediated somatic growth that occurs during childhood andadolescence is dependent on the endocrine form of IGF-1 that is mainlyproduced and secreted by the liver (Daughaday, 2000, PediatricNephrology 14: 537-40). The synthesis of hepatic IGF-1 is stimulated byGH release in the pituitary in response to hypothalamic GHRH (GHreleasing hormone). The serum concentration of IGF-1 increases over 100fold between ages 5-15 in humans. The bioavailability of IGF-1 isregulated by IGF binding protein 3 (IGFBP3) with approximately 99% ofthe growth factor compartmentalized in the bound state. Primary IGF-1deficiency arising form partial gene deletions, and secondary IGF-1deficiency resulting from defects in GH production or signaling are notlethal (Woods, 1999, IGF Deficiency in Contemporary Endocrinology: TheIGF System, R. a. R. Rosenfeld, C. Jr. Totowa, eds., Humana Press, N.J.:651-74). The affected individuals exhibit growth retardation at birth,grow slowly and can face certain CNS abnormalities.

IGF-1R signaling promotes cell growth and survival through the IRSadapter protein-dependent activation of the PI3Kinase/Akt pathway.IGF-1R transmits a signal to its major substrates, IRS-1 through IRS-4and the Shc proteins (Blakesley et al., 1999, IGF-1 receptor function:transducing the IGF-1 signal into intracellular events in The IGFSystem, R. G. a. R. Rosenfeld, Jr. C. T. Totowa, eds., Humana Press,N.J.: 143-63). This results in activation of the Ras/Raf/MAP kinase andPI3 Kinase/Akt signaling pathways. However, induction of Akt-mediatedcell survival via IRS is the dominant pathway response upon IGFstimulation of most cells. See FIG. 10.

Antigen Binding Proteins

In one aspect, the present invention provides antigen binding proteins(e.g., antibodies, antibody fragments, antibody derivatives, antibodymuteins, and antibody variants), that bind to IGF-1R, e.g., humanIGF-1R.

Antigen binding proteins in accordance with the present inventioninclude antigen binding proteins that inhibit a biological activity ofIGF-1R. Examples of such biological activities include binding asignaling molecule (e.g., IGF-1 and/or IGF-2), and transducing a signalin response to binding a signaling molecule.

Different antigen binding proteins may bind to different domains orepitopes of IGF-1R or act by different mechanisms of action. Examplesinclude but are not limited to antigen binding proteins that interferewith binding of IGF-1 and/or IGF-2 to IGF-1R or that inhibit signaltransduction. The site of action may be, for example, intracellular(e.g., by interfering with an intracellular signaling cascade) orextracellular. An antigen binding protein need not completely inhibit anIGF-1 and/or IGF-2 induced activity to find use in the presentinvention; rather, antigen binding proteins that reduce a particularactivity of IGF-1 and/or IGF-2 are contemplated for use as well.(Discussions herein of particular mechanisms of action forIGF-1R-binding antigen binding proteins in treating particular diseasesare illustrative only, and the methods presented herein are not boundthereby.)

It has been observed that IGF-1 and IGF-2 each exhibits biphasic bindingto IGF-1R. High affinity binding has been reported to have a K_(D) inthe range of 0.2 nM; high affinity binding, about ten fold higher. Thus,in one embodiment, the present invention provides an IGF-1R inhibitorthat inhibits both the high and low affinity binding of IGF-1 and/orIGF-2 to IGF-R. It has been suggested that the high affinity binding,rather than the low affinity binding, of IGF-1 and/or IGF-2 to IGF-1R isrequired for the conformation change that activates the tyrosine kinaseactivity of IGF-1R. Thus, in another embodiment, the IGF-1R inhibitorpreferentially inhibits the high affinity binding of IGF-1 and/or IGF-2to IGF-1R as compared to the low affinity binding.

In another aspect, the present invention provides antigen bindingproteins that comprise a light chain variable region selected from thegroup consisting of L1 through L52 and/or a heavy chain variable regionselected from the group consisting of H1 through H52, and fragments,derivatives, muteins, and variants thereof (see FIGS. 2 and 3). Such anantigen binding protein can be denoted using the nomenclature “LxHy”,wherein “x” corresponds to the number of the light chain variable regionand “y” corresponds to the number of the heavy chain variable region asthey are labeled in FIGS. 2 and 3. For example, L2H1 refers to anantigen binding protein with a light chain variable region comprisingthe amino acid sequence of L2 and a heavy chain variable regioncomprising the amino acid sequence of H1, as shown in FIGS. 2 and 3.FIGS. 2 and 3 also indicate the location of the CDR and frameworkregions of each of these variable domain sequences. The CDR regions ofeach light and heavy chain also are grouped by type and by sequencesimilarity in FIGS. 4 through 9. Antigen binding proteins of theinvention include, for example, antigen binding proteins having acombination of light chain and heavy chain variable domains selectedfrom the group of combinations consisting of L1H1, L2H2, L3H3, L4H4,L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14,L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23,L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31, L32H32,L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41,L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50,L51H51, and L52H52.

In one embodiment, the present invention provides an antigen bindingprotein comprising a light chain variable domain comprising a sequenceof amino acids that differs from the sequence of a light chain variabledomain selected from the group consisting of L1 through L52 only at 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residues, wherein eachsuch sequence difference is independently either a deletion, insertion,or substitution of one amino acid residue. In another embodiment, thelight-chain variable domain comprises a sequence of amino acids that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to thesequence of a light chain variable domain selected from the groupconsisting of L1 through L52. In another embodiment, the light chainvariable domain comprises a sequence of amino acids that is encoded by anucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%,or 99% identical to a nucleotide sequence that encodes a light chainvariable domain selected from the group consisting of L1 through L52. Inanother embodiment, the light chain variable domain comprises a sequenceof amino acids that is encoded by a polynucleotide that hybridizes undermoderately stringent conditions to the complement of a polynucleotidethat encodes a light chain variable domain selected from the groupconsisting of L1 through L52. In another embodiment, the light chainvariable domain comprises a sequence of amino acids that is encoded by apolynucleotide that hybridizes under moderately stringent conditions tothe complement of a polynucleotide that encodes a light chain variabledomain selected from the group consisting of L1 through L52. In anotherembodiment, the light chain variable domain comprises a sequence ofamino acids that is encoded by a polynucleotide that hybridizes undermoderately stringent conditions to a complement of a light chainpolynucleotide selected from FIG. 1.

In another embodiment, the present invention provides an antigen bindingprotein comprising a heavy chain variable domain comprising a sequenceof amino acids that differs from the sequence of a heavy chain variabledomain selected from the group consisting of H1 through H52 only at 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue(s), whereineach such sequence difference is independently either a deletion,insertion, or substitution of one amino acid residue. In anotherembodiment, the heavy chain variable domain comprises a sequence ofamino acids that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%identical to the sequence of a heavy chain variable domain selected fromthe group consisting of H1 through H52. In another embodiment, the heavychain variable domain comprises a sequence of amino acids that isencoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%,90%, 95%, 97%, or 99% identical to a nucleotide sequence that encodes aheavy chain variable domain selected from the group consisting of H1through H52. In another embodiment, the heavy chain variable domaincomprises a sequence of amino acids that is encoded by a polynucleotidethat hybridizes under moderately stringent conditions to the complementof a polynucleotide that encodes a heavy chain variable domain selectedfrom the group consisting of H1 through H52. In another embodiment, theheavy chain variable domain comprises a sequence of amino acids that isencoded by a polynucleotide that hybridizes under moderately stringentconditions to the complement of a polynucleotide that encodes a heavychain variable domain selected from the group consisting of H1 throughH52. In another embodiment, the heavy chain variable domain comprises asequence of amino acids that is encoded by a polynucleotide thathybridizes under moderately stringent conditions to a complement of aheavy chain polynucleotide selected from FIG. 1.

Particular embodiments of antigen binding proteins of the presentinvention comprise one or more amino acid sequences that are identicalto the amino acid sequences of one or more of the CDRs and/or FRsillustrated in FIGS. 2 through 9. In one embodiment, the antigen bindingprotein comprises a light chain CDR1 sequence illustrated in FIG. 4. Inanother embodiment, the antigen binding protein comprises a light chainCDR2 sequence illustrated in FIG. 5. In another embodiment, the antigenbinding protein comprises a light chain CDR3 sequence illustrated inFIG. 6. In another embodiment, the antigen binding protein comprises aheavy chain CDR1 sequence illustrated in FIG. 7. In another embodiment,the antigen binding protein comprises a heavy chain CDR2 sequenceillustrated in FIG. 8. In another embodiment, the antigen bindingprotein comprises a heavy chain CDR3 sequence illustrated in FIG. 9. Inanother embodiment, the antigen binding protein comprises a light chainFR1 sequence illustrated in FIG. 2. In another embodiment, the antigenbinding protein comprises a light chain FR2 sequence illustrated in FIG.2. In another embodiment, the antigen binding protein comprises a lightchain FR3 sequence illustrated in FIG. 2. In another embodiment, theantigen binding protein comprises a light chain FR4 sequence illustratedin FIG. 2. In another embodiment, the antigen binding protein comprisesa heavy chain FR1 sequence illustrated in FIG. 3. In another embodiment,the antigen binding protein comprises a heavy chain FR2 sequenceillustrated in FIG. 3. In another embodiment, the antigen bindingprotein comprises a heavy chain FR3 sequence illustrated in FIG. 3. Inanother embodiment, the antigen binding protein comprises a heavy chainFR4 sequence illustrated in FIG. 3.

In one embodiment, the present invention provides an antigen bindingprotein that comprises one or more CDR sequences that differ from a CDRsequence shown in FIGS. 2 through 9 by no more than 5, 4, 3, 2, or 1amino acid residues.

In one embodiment, the present invention provides an antigen bindingprotein that comprises at least one CDR from L1-L52 and/or H1-H52, asshown in FIGS. 2 through 9, and at least one CDR sequence from ananti-IGF-1R antibody described in US Pat. App. Pub. Nos. 03/0235582,04/0228859, 04/0265307, 04/0886503, 05/0008642, 05/0084906, 05/0186203,05/0244408, PCT Pub. Nos. WO 03/059951, WO 03/100008, WO 04/071529A2, WO04/083248, WO 04/087756, WO 05/016967, WO 05/016970, or WO 05/058967(each of which is incorporated herein by reference in its entirety forall purposes) wherein the antigen binding protein binds to IGF-1receptor. In another embodiment, the antigen binding protein comprises2, 3, 4, or 5 CDR sequences from L1-L52 and/or H1-H52, as shown in FIGS.2 through 9. In another embodiment, the antigen binding proteincomprises 2, 3, 4, or 5 CDR sequences from an anti-IGF-1R antibodydescribed in US Pat. App. Pub. Nos. 03/0235582, 04/0228859, 04/0265307,04/0886503, 05/0008642, 05/0084906, 05/0186203, 05/0244408, PCT Pub.Nos. WO 03/059951, WO 03/100008, WO 04/071529A2, WO 04/083248, WO04/087756, WO 05/016967, WO 05/016970, or WO 05/058967. In anotherembodiment, at least one of the antigen binding protein's CDR3 sequencesis a CDR3 sequence from L1-L52 and/or H1-H52, as shown in FIGS. 2, 3, 6,and 9. In another embodiment, the antigen binding protein's light chainCDR3 sequence is a light chain CDR3 sequence from L1-L52 as shown inFIGS. 2 and 6 and the antigen binding protein's heavy chain CDR3sequence is a heavy chain sequence from H1-H52 as shown in FIGS. 3 and9. In another embodiment, the antigen binding protein comprises 1, 2, 3,4, or 5 CDR sequences that each independently differs by 6, 5, 4, 3, 2,1, or 0 single amino acid additions, substitutions, and/or deletionsfrom a CDR sequence of L1-L52 and/or H1-H52, and the antigen bindingprotein further comprises 1, 2, 3, 4, or 5 CDR sequences that eachindependently differs by 6, 5, 4, 3, 2, 1, or 0 single amino acidadditions, substitutions, and/or deletions from a CDR sequence of USPat. App. Pub. Nos. 03/0235582, 04/0228859, 04/0265307, 04/0886503,05/0008642, 05/0084906, 05/0186203, 05/0244408, PCT Pub. Nos. WO03/059951, WO 03/100008, WO 04/071529A2, WO 04/083248, WO 04/087756, WO05/016967, WO 05/016970, or WO 05/058967. In another embodiment, the CDRsequence(s) from US Pat. App. Pub. Nos. 03/0235582, 04/0228859,04/0265307, 04/0886503, 05/0008642, 05/0084906, 05/0186203, 05/0244408,PCT Pub. Nos. WO 03/059951, WO 03/100008, WO 04/071529A2, WO 04/083248,WO 04/087756, WO 05/016967, WO 05/016970, or WO 05/058967. In anotherembodiment, the CDR sequence(s) are from (an) antibody(-ies) thatbind(s) to the L2 portion of the extracellular domain of IGF-1 receptor.In another embodiment, the antigen binding protein does not comprise alight chain CDR3 sequence and/or a heavy chain CDR3 sequence from ananti-IGF-1R antibody from US Pat. App. Pub. Nos. 03/0235582, 04/0228859,04/0265307, 04/0886503, 05/0008642, 05/0084906, 05/0186203, 05/0244408,PCT Pub. Nos. WO 03/059951, WO 03/100008, WO 04/071529A2, WO 04/083248,WO 04/087756, WO 05/016967, WO 05/016970, or WO 05/058967.

In one embodiment, the present invention provides an antigen bindingprotein that comprises a light chain CDR1 comprising the sequenceRSSQSLLHX₁X₂GYNX₃LX₄ (SEQ ID NO:236), wherein X₁ is a serine or athreonine residue, X₂ is an asparagine, serine, or histidine residue, X₃is a tyrosine or a phenylalanine residue, and X₄ is an aspartate or anasparagine residue. In another embodiment, the light chain CDR1comprises the sequence TRSSGX₁IX₂X₃NYVQ (SEQ ID NO:237), wherein X₁ is aserine or an aspartate residue, X₂ is an alanine or an aspartateresidue, and X₃ is a serine or an asparagine residue. In anotherembodiment, the light chain CDR1 comprises the sequenceRASQX₁X₂X₃X₄X₅LX₆ (SEQ ID NO:238), wherein X₁ is a glycine or a serineresidue, X₂ is an isoleucine, valine, or proline residue, and X₃ is aserine, glycine, or tyrosine residue, X₄ is any amino acid residue, X₅is a phenylalanine, tyrosine, asparagine, or tryptophan residue, and X₆is an alanine or an asparagine residue. In another embodiment, X₂ is anisoleucine or valine residue, X₃ is a glycine or serine residue, X₄ isan arginine, serine, asparagine, serine, tyrosine, or isoleucineresidue, and X₅ is a phenylalanine or a tyrosine residue.

In one embodiment, the present invention provides an antigen bindingprotein that comprises a light chain CDR2 comprising the sequenceLX₁X₂X₃RX₄S (SEQ ID NO:239), wherein X₁ is a glycine or a valineresidue, X₂ is a serine or a phenylalanine residue, X₃ is an asparagine,tyrosine, or threonine residue, and X₄ is an alanine or an aspartateresidue. In another embodiment, the CDR2 comprises the sequenceAX₁SX₂LX₃S (SEQ ID NO:240), wherein X₁ is an alanine or a threonineresidue, X₂ is a threonine or a glycine residue, and X₃ is a glutamineor a glutamate residue. In another embodiment, the CDR2 comprises thesequence X₁X₂NX₃RPS (SEQ ID NO:241), wherein X₁ is a glutamate,glutamine, or glycine residue, X₂ is an aspartate or lysine residue, andX₃ is any amino acid residue.

In one embodiment, the present invention provides an antigen bindingprotein that comprises a light chain CDR3 comprising the sequenceMX₁X₂X₃X₄X₅PX₆X₇ (SEQ ID NO:242), wherein X₁ is a glutamine or glutamateresidue, X₂ is an alanine, glycine, serine, or threonine residue, X₃ isa leucine or threonine residue, X₄ is a glutamine, glutamate, orhistidine residue, X₅ is a threonine, tryptophan, methionine, or valineresidue, X₆ is a nonpolar side chain residue, and X₇ is a threonine,serine, or alanine residue. In another embodiment, the CDR3 comprisesthe sequence QQX₁X₂X₃X₄PX₅T (SEQ ID NO:243), wherein X₁ is an arginine,serine, leucine, or alanine residue, X₂ is an asparagine, serine, orhistidine residue, X₃ is a serine or an asparagine residue, X₄ is anonpolar side chain residue, and X₅ is a leucine, isoleucine, tyrosine,or tryptophan residue. In another embodiment, the CDR3 comprises thesequence QSYX₁SX₂NX₃X₄V (SEQ ID NO:244), wherein X₁ is an aspartate or aglutamine residue, X₂ is a serine or an aspartate residue, X₃ is aglutamine, valine, or tryptophan residue, and X₄ is an arginine residueor no residue.

In one embodiment, the present invention provides an antigen bindingprotein that comprises a heavy chain CDR1 comprising the sequenceX₁X₂X₃WWS (SEQ ID NO:245), wherein X₁ is a serine residue or no residue,X₂ is a serine or asparagine residue, and X₃ is an asparagine residueand an isoleucine residue. In another embodiment, the heavy chain CDR1comprises the sequence X₁X₂YWS (SEQ ID NO:246), wherein X₁ is a glycine,asparagine, or aspartate residue, and X₂ is a tyrosine or phenylalanineresidue. In another embodiment, the heavy chain CDR1 comprises thesequence SYX₁X₂X₃ (SEQ ID NO:247), wherein X₁ is an alanine or glycineresidue, X₂ is a methionine or isoleucine residue, and X₃ is a serine orhistidine residue.

In one embodiment, the present invention provides an antigen bindingprotein that comprises a heavy chain CDR2 comprising the sequenceX₁X₂X₃X₄X₅GX₆TXANPSLX₈S (SEQ ID NO:248), wherein X₁ is a glutamate,tyrosine, or serine residue, X₂ is a isoleucine or valine residue, X₃ isa tyrosine, asparagine, or serine residue, X₄ is a histidine, tyrosine,aspartate, or proline residue, X₅ is a serine or arginine residue, X₆ isa serine or asparagine residue, X₇ is an asparagine or tyrosine residue,and X₈ is a lysine or glutamate residue. In another embodiment, theheavy chain CDR2 comprises the sequence X₁ISX₂X₃X₄X₅X₆X₇YYADSVKG (SEQ IDNO:249), wherein X₁ is a threonine, alanine, valine, or tyrosineresidue, X₂ is a glycine, serine, or tyrosine residue, X₃ is a serine,asparagine, or aspartate residue, X₄ is a glycine or serine residue, X₅is a glycine, serine, or aspartate residue, X₆ is a serine, threonine,or asparagine residue, and X₇ is a threonine, lysine, or isoleucineresidue.

In one embodiment, the present invention provides an antigen bindingprotein that comprises a heavy chain CDR3 comprising the sequenceX₁X₂X₃X₄X₅X₆X₇X₈X₉FDI (SEQ ID NO:250), wherein X₁ is a glutamate residueor no residue, X₂ is tyrosine, glycine, or serine residue or no residue,X₃ is a serine, asparagine, tryptophan, or glutamate residue, or noresidue, X₄ is a serine, aspartate, tryptophan, alanine, arginine,threonine, glutamine, leucine, or glutamate residue, or no residue, X₅is a serine, glycine, asparagine, threonine, tryptophan, alanine,valine, or isoleucine residue, X₆ is an arginine, glutamine, tyrosine,valine, alanine, glycine, serine, phenylalanine, or tryptophan residue,X₇ is a leucine, asparagine, aspartate, threonine, tryptophan, tyrosine,valine, alanine, or histidine residue, X₈ is an aspartate, serine,asparagine, or glutamine residue, and X₉ is an alanine or a prolineresidue. In another embodiment, the heavy chain CDR3 comprises thesequence X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁MDV (SEQ ID NO:251), wherein X₁ is analanine residue, or no residue, X₂ is a glutamate, tyrosine, or glycineresidue, or no residue, X₃ is a serine or arginine residue, or noresidue, X₄ is an aspartate, glycine, serine, or valine residue, or noresidue, X₅ is a serine, glycine, or aspartate residue, or no residue,X₆ is a glycine, phenylalanine, aspartate, serine, tryptophan, ortyrosine residue, or no residue, X₇ is a tyrosine, tryptophan, serine,or aspartate residue, or no residue, X₈ is an aspartate, arginine,serine, glycine, tyrosine, or tryptophan residue, X₉ is a tyrosine,isoleucine, leucine, phenylalanine, or lysine residue, X₁₀ is atyrosine, phenylalanine, aspartate, or glycine residue, and X₁₁ is aglycine, tyrosine, or asparagine residue. In another embodiment, theheavy chain CDR3 comprises the sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀Y (SEQ IDNO:252), wherein X₁ is an aspartate or valine residue, or no residue, X₂is a glycine, tyrosine, arginine, or aspartate residue, or no residue,X₃ is an asparagine, leucine, glycine, isoleucine, serine, valine,phenylalanine, or tyrosine residue, or no residue, X₄ is a leucine,serine, tryptophan, alanine, tyrosine, isoleucine, glycine, or aspartateresidue, or no residue, X₅ is a glycine, alanine, tyrosine, serine,aspartate, or leucine residue, X₆ is a valine, alanine, glycine,threonine, proline, histidine, or glutamine residue, X₇ is a glutamate,glycine, serine, aspartate, glycine, valine, tryptophan, histidine, orarginine residue, X₈ is a glutamine, alanine, glycine, tyrosine,proline, leucine, aspartate, or serine residue, X₉ is a nonpolar sidechain residue, and X₁₀ is an aspartate or alanine residue. In anotherembodiment, the heavy chain CDR3 comprises the sequenceX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀YFDX₁₁ (SEQ ID NO:253), wherein X₁ is a glycineresidue, or no residue, X₂ is a proline residue, or no residue, X₃ is anarginine or aspartate residue, or no residue, X₄ is a histidine orproline residue, X₅ is an arginine or glycine residue, X₆ is anarginine, serine, or phenylalanine residue, X₇ is an aspartate or serineresidue, X₈ is a glycine, tryptophan, or tyrosine residue, X₉ is atyrosine or alanine residue, X₁₀ is an asparagine or tryptophan residue,and X₁₁ is an asparagine or leucine residue. In another embodiment, theheavy chain CDR3 comprises the sequence X₁X₂X₃X₄DSSX₅X₆X₇X₈X₉X₁₀X₁₁X₁₂(SEQ ID NO:254), wherein X₁ is a phenylalanine residue, or no residue,X₂ is an asparagine or glycine residue, or no residue, X₃ is a tyrosineor a leucine residue, or no residue, X₄ is a tyrosine or glycineresidue, or no residue, X₅ is a glycine, serine, or valine residue, X₆is a tyrosine, phenylalanine, tryptophan, or glutamine residue, or noresidue, X₇ is a tyrosine, glycine, or isoleucine residue, or noresidue, X₈ is a tyrosine, leucine, or glycine residue, or no residue,X₉ is a methionine, glycine, or phenylalanine residue, or no residue,X₁₀ is an aspartate or methionine residue, or no residue, X₁₁ is avaline, aspartate, or tyrosine residue, or no residue, and X₁₂ is avaline residue, or no residue.

In one embodiment, the present invention provides an isolated antigenbinding protein, comprising either: a. a light chain CDR3 comprising asequence selected from the group consisting of: i. a light chain CDR3sequence selected from the group consisting of the light chain CDR3sequences of L1-L52 as shown in FIG. 6; ii. MQALQTPZT; iii.QQ(R/S)(N/S)(S/N)ZPLT; and iv. QSYDSSNXJV; b. a heavy chain CDR3comprising a sequence selected from the group consisting of: i. a heavychain CDR3 sequence that differs by no more than a total of three aminoacid additions, substitutions, or deletions from a CDR3 sequenceselected from the group consisting of the heavy chain CDR3 sequences ofH1-H52 as shown in FIG. 9; ii. SRLDAFDI; iii. SXYDYYGMDV; iv.HRXDXAWYFDL; and v. DSSG; or c. the light chain CDR3 sequence of (a) andthe heavy chain CDR3 sequence of (b); wherein amino acid residue symbolsenclosed in parentheses identify alternative residues for the sameposition in a sequence, each X is independently any amino acid residue,each Z is independently a glycine residue, an alanine residue, a valineresidue, a leucine residue, an isoleucine residue, a proline residue, aphenylalanine residue, a methionine residue, a tryptophan residue, or acysteine residue, each J is independently a glutamine residue, anarginine residue, a valine residue, or a tryptophan residue, and theantigen binding protein binds to human IGF-1R.

The nucleotide sequences of FIG. 1, or the amino acid sequences of FIGS.2 through 9, can be altered, for example, by random mutagenesis or bysite-directed mutagenesis (e.g., oligonucleotide-directed site-specificmutagenesis) to create an altered polynucleotide comprising one or moreparticular nucleotide substitutions, deletions, or insertions ascompared to the non-mutated polynucleotide. Examples of techniques formaking such alterations are described in Walder et al., 1986, Gene42:133; Bauer et al. 1985, Gene 37:73; Craik, BioTechniques, January1985, 12-19; Smith et al., 1981, Genetic Engineering: Principles andMethods, Plenum Press; and U.S. Pat. Nos. 4,518,584 and 4,737,462. Theseand other methods can be used to make, for example, derivatives ofanti-IGF-1R antibodies that have a desired property, for example,increased affinity, avidity, or specificity for IGF-1R, increasedactivity or stability in vivo or in vitro, or reduced in vivoside-effects as compared to the underivatized antibody.

Other derivatives of anti-IGF-1R antibodies within the scope of thisinvention include covalent or aggregative conjugates of anti-IGF-1Rantibodies, or fragments thereof, with other proteins or polypeptides,such as by expression of recombinant fusion proteins comprisingheterologous polypeptides fused to the N-terminus or C-terminus of ananti-IGF-1R antibody polypeptide. For example, the conjugated peptidemay be a heterologous signal (or leader) polypeptide, e.g., the yeastalpha-factor leader, or a peptide such as an epitope tag. Antigenbinding protein-containing fusion proteins can comprise peptides addedto facilitate purification or identification of antigen binding protein(e.g., poly-His). An antigen binding protein also can be linked to theFLAG peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:255)as described in Hopp et al., Bio/Technology 6:1204, 1988, and U.S. Pat.No. 5,011,912. The FLAG peptide is highly antigenic and provides anepitope reversibly bound by a specific monoclonal antibody (mAb),enabling rapid assay and facile purification of expressed recombinantprotein. Reagents useful for preparing fusion proteins in which the FLAGpeptide is fused to a given polypeptide are commercially available(Sigma, St. Louis, Mo.).

Oligomers that contain one or more antigen binding proteins may beemployed as IGF-1R antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to theantigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have IGF-1R binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature344:677; and Hollenbaugh et al., 1992 “Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11.

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing an IGF-1R bindingfragment of an anti-IGF-1R antibody to the Fc region of an antibody. Thedimer can be made by, for example, inserting a gene fusion encoding thefusion protein into an appropriate expression vector, expressing thegene fusion in host cells transformed with the recombinant expressionvector, and allowing the expressed fusion protein to assemble much likeantibody molecules, whereupon interchain disulfide bonds form betweenthe Fc moieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG1 antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein isidentical to that of the native Fc sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of an anti-IGF-1R antibody may be substituted for the variableportion of an antibody heavy and/or light chain.

Alternatively, the oligomer is a fusion protein comprising multipleantigen binding proteins, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT applicationWO 94/10308, and the leucine zipper derived from lung surfactant proteinD (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In oneapproach, recombinant fusion proteins comprising an anti-IGF-1R antibodyfragment or derivative fused to a leucine zipper peptide are expressedin suitable host cells, and the soluble oligomeric anti-IGF-1R antibodyfragments or derivatives that form are recovered from the culturesupernatant.

In one aspect, the present invention provides antigen binding proteinsthat interfere with the binding of IGF-1 and/or IGF-2 to an IGF-1R. Suchantigen binding proteins can be made against IGF-1R, or a fragment,variant or derivative thereof, and screened in conventional assays forthe ability to interfere with binding of IGF-1 and/or IGF-2 to IGF-1R.Examples of suitable assays are assays that test the antigen bindingproteins for the ability to inhibit binding of IGF-1 and/or IGF-2 tocells expressing IGF-1R, or that test antigen binding proteins for theability to reduce a biological or cellular response that results fromthe binding of IGF-1 and/or IGF-2 to cell surface IGF-1R receptors.

In another aspect, the present invention provides an antigen bindingprotein that blocks the binding of IGF-1 and/or IGF-2 to IGF-1R but doesnot significantly block the binding of insulin to insulin receptor(INS-R). In one embodiment, the antigen binding protein does not bind toINS-R. In another embodiment, the antigen binding protein binds to theINS-R with such a low affinity that it does not effectively block thebinding of insulin to INS-R. In another embodiment, the antigen bindingprotein binds to INS-R, but antigen binding protein-bound INS-R canstill bind to insulin. In another embodiment, the antigen bindingprotein's selectivity for IGF-1R is at least 50 times greater than itsselectivity for insulin receptor. In another embodiment, the selectivityof the antigen binding protein is more than 100 times greater than itsselectivity for insulin receptor.

In another aspect, the present invention provides an antigen bindingprotein that demonstrates species selectivity. In one embodiment, theantigen binding protein binds to one or more mammalian IGF-1R, forexample, to human IGF-1R and one or more of mouse, rat, guinea pig,hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, andnon-human primate IGF-1R. In another embodiment, the antigen bindingprotein binds to one or more primate IGF-1R, for example, to humanIGF-1R and one or more of cynomologous, marmoset, rhesus, and chimpanzeeIGF-1R. In another embodiment, the antigen binding protein bindsspecifically to human, cynomologous, marmoset, rhesus, or chimpanzeeIGF-1R. In another embodiment, the antigen binding protein does not bindto one or more of mouse, rat, guinea pig, hamster, gerbil, cat, rabbit,dog, goat, sheep, cow, horse, camel, and non-human primate IGF-1R. Inanother embodiment, the antigen binding protein does not bind to a NewWorld monkey species such as a marmoset. In another embodiment, theantigen binding protein does not exhibit specific binding to anynaturally occurring protein other than IGF-1R. In another embodiment,the antigen binding protein does not exhibit specific binding to anynaturally occurring protein other than mammalian IGF-1R. In anotherembodiment, the antigen binding protein does not exhibit specificbinding to any naturally occurring protein other than primate IGF-1R. Inanother embodiment, the antigen binding protein does not exhibitspecific binding to any naturally occurring protein other than humanIGF-1R. In another embodiment, the antigen binding protein specificallybinds to mouse, rat, cynomolgus monkey, and human IGF-1R. In anotherembodiment, the antigen binding protein specifically binds to mouse,rat, cynomolgus monkey, and human IGF-1R with a similar bindingaffinity. In another embodiment, the antigen binding protein blocksbinding of human IGF-1 and IGF-2 with mouse, rat, cynomolgus monkey, andhuman IGF-1R. In another embodiment, the antigen binding protein blocksbinding of human IGF-1 and IGF-2 with mouse, rat, cynomolgus monkey, andhuman IGF-1R with similar K_(i). In another embodiment, the antigenbinding protein blocks binding of human IGF-1 and IGF-2 with mouse, rat,cynomolgus monkey, and human IGF-1R with a K₁ of between about 0.57 andabout 0.61 nM.

One may determine the selectivity of an antigen binding protein for anIGF-1R using methods well known in the art and following the teachingsof the specification. For example, one may determine the selectivityusing Western blot, FACS, ELISA or RIA.

In another aspect, the present invention provides an IGF-1R bindingantigen binding protein (for example, an anti-IGF-1R antibody), that hasone or more of the following characteristics: binds to both human andmurine IGF-1R, inhibits the binding of both IGF-1 and IGF-2 to humanIGF-1R, inhibits the binding of both IGF-1 and IGF-2 to murine IGF-1R,preferentially inhibits the high affinity binding of IGF-1 and/or ofIGF-2 to IGF-1R, binds to the L2 domain of IGF-1R, causes relativelylittle down-regulation of cell-surface expressed IGF-1R after 17 hoursof exposure (as compared to MAB391 (R&D systems, Minneapolis, Minn.);e.g., amount of IGF-1R is reduced by less than 20%), causes a level ofdown-regulation of cell-surface expressed IGF-1R on Colo-205 orMiaPaCa-2 xenograft tumor cells in mice as MAB391 after four weeks ofonce weekly doses of 200 micrograms.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)₂ fragments. Antibodyfragments and derivatives produced by genetic engineering techniquesalso are contemplated.

Additional embodiments include chimeric antibodies, e.g., humanizedversions of non-human (e.g., murine) monoclonal antibodies. Suchhumanized antibodies may be prepared by known techniques, and offer theadvantage of reduced immunogenicity when the antibodies are administeredto humans. In one embodiment, a humanized monoclonal antibody comprisesthe variable domain of a murine antibody (or all or part of the antigenbinding site thereof) and a constant domain derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variabledomain fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal., 1988, Nature 332:323, Liu et al., 1987, Proc. Nat. Acad. Sci. USA84:3439, Larrick et al., 1989, Bio/Technology 7:934, and Winter et al.,1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDRgrafted antibody. Techniques for humanizing antibodies are discussed in,e.g., U.S. patent application Ser. No. 10/194,975 (published Feb. 27,2003), U.S. Pat. Nos. 5,869,619, 5,225,539, 5,821,337, 5,859,205, Padlanet al., 1995, FASEB J. 9:133-39, and Tamura et al., 2000, J. Immunol.164:1432-41.

Procedures have been developed for generating human or partially humanantibodies in non-human animals. For example, mice in which one or moreendogenous immunoglobulin genes have been inactivated by various meanshave been prepared. Human immunoglobulin genes have been introduced intothe mice to replace the inactivated mouse genes. Antibodies produced inthe animal incorporate human immunoglobulin polypeptide chains encodedby the human genetic material introduced into the animal. In oneembodiment, a non-human animal, such as a transgenic mouse, is immunizedwith an IGF-1R polypeptide, such that antibodies directed against theIGF-1R polypeptide are generated in the animal. One example of asuitable immunogen is a soluble human IGF-1R, such as a polypeptidecomprising the extracellular domain of the protein of FIG. 10, or otherimmunogenic fragment of the protein of FIG. 10. Examples of techniquesfor production and use of transgenic animals for the production of humanor partially human antibodies are described in U.S. Pat. Nos. 5,814,318,5,569,825, and 5,545,806, Davis et al., 2003, Production of humanantibodies from transgenic mice in Lo, ed. Antibody Engineering: Methodsand Protocols, Humana Press, N.J.:191-200, Kellermann et al., 2002, CurrOpin Biotechnol. 13:593-97, Russel et al., 2000, Infect Immun.68:1820-26, Gallo et al., 2000, Eur J Immun. 30:534-40, Davis et al.,1999, Cancer Metastasis Rev. 18:421-25, Green, 1999, J Immunol Methods.231:11-23, Jakobovits, 1998, Advanced Drug Delivery Reviews 31:33-42,Green et al., 1998, J Exp Med. 188:483-95, Jakobovits A, 1998, Exp.Opin. Invest. Drugs. 7:607-14, Tsuda et al., 1997, Genomics. 42:413-21,Mendez et al., 1997, Nat Genet. 15:146-56, Jakobovits, 1994, Curr Biol.4:761-63, Arbones et al., 1994, Immunity. 1:247-60, Green et al., 1994,Nat Genet. 7:13-21, Jakobovits et al., 1993, Nature. 362:255-58,Jakobovits et al., 1993, Proc Natl Acad Sci USA. 90:2551-55. Chen, J.,M. Trounstine, F. W. Alt, F. Young, C. Kurahara, J. Loring, D. Huszar.“Immunoglobulin gene rearrangement in B cell deficient mice generated bytargeted deletion of the JH locus.” International Immunology 5 (1993):647-656, Choi et al., 1993, Nature Genetics 4: 117-23, Fishwild et al.,1996, Nature Biotechnology 14: 845-51, Harding et al., 1995, Annals ofthe New York Academy of Sciences, Lonberg et al., 1994, Nature 368:856-59, Lonberg, 1994, Transgenic Approaches to Human MonoclonalAntibodies in Handbook of Experimental Pharmacology 113: 49-101, Lonberget al., 1995, Internal Review of Immunology 13: 65-93, Neuberger, 1996,Nature Biotechnology 14: 826, Taylor et al., 1992, Nucleic AcidsResearch 20: 6287-95, Taylor et al., 1994, International Immunology 6:579-91, Tomizuka et al., 1997, Nature Genetics 16: 133-43, Tomizuka etal., 2000, Proceedings of the National Academy of Sciences USA 97:722-27, Tuaillon et al., 1993, Proceedings of the National Academy ofSciences USA 90: 3720-24, and Tuaillon et al., 1994, Journal ofImmunology 152: 2912-20.

In another aspect, the present invention provides monoclonal antibodiesthat bind to IGF-1R. Monoclonal antibodies may be produced using anytechnique known in the art, e.g., by immortalizing spleen cellsharvested from the transgenic animal after completion of theimmunization schedule. The spleen cells can be immortalized using anytechnique known in the art, e.g., by fusing them with myeloma cells toproduce hybridomas. Myeloma cells for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Examples of suitable cell lines foruse in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions areU-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

In one embodiment, a hybridoma cell line is produced by immunizing ananimal (e.g., a transgenic animal having human immunoglobulin sequences)with an IGF-1R immunogen; harvesting spleen cells from the immunizedanimal; fusing the harvested spleen cells to a myeloma cell line,thereby generating hybridoma cells; establishing hybridoma cell linesfrom the hybridoma cells, and identifying a hybridoma cell line thatproduces an antibody that binds an IGF-1R polypeptide. Such hybridomacell lines, and anti-IGF-1R monoclonal antibodies produced by them, areencompassed by the present invention.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any technique known in the art. Hybridomas or mAbs may be furtherscreened to identify mAbs with particular properties, such as theability to block an IGF-1 and/or IGF-2 induced activity. Examples ofsuch screens are provided in the examples below.

Molecular evolution of the complementarity determining regions (CDRs) inthe center of the antibody binding site also has been used to isolateantibodies with increased affinity, for example, antibodies havingincreased affinity for c-erbB-2, as described by Schier et al., 1996, J.Mol. Biol. 263:551. Accordingly, such techniques are useful in preparingantibodies to IGF-1R.

Antigen binding proteins directed against an IGF-1R can be used, forexample, in assays to detect the presence of IGF-1R polypeptides, eitherin vitro or in vivo. The antigen binding proteins also may be employedin purifying IGF-1R proteins by immunoaffinity chromatography. Thoseantigen binding proteins that additionally can block binding of IGF-1and/or IGF-2 to IGF-1R may be used to inhibit a biological activity thatresults from such binding. Blocking antigen binding proteins can be usedin the methods of the present invention. Such antigen binding proteinsthat function as IGF-1 and/or IGF-2 antagonists may be employed intreating any IGF-1 and/or IGF-2-induced condition, including but notlimited to cancer. In one embodiment, a human anti-IGF-1R monoclonalantibody generated by procedures involving immunization of transgenicmice is employed in treating such conditions.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit an IGF-1 and/or IGF-2-induced biologicalactivity. Disorders caused or exacerbated (directly or indirectly) bythe interaction of IGF-1 and/or IGF-2 with cell surface IGF-1R, examplesof which are provided above, thus may be treated. In one embodiment, thepresent invention provides a therapeutic method comprising in vivoadministration of an IGF-1 and/or IGF-2 blocking antigen binding proteinto a mammal in need thereof in an amount effective for reducing an IGF-1and/or IGF-2-induced biological activity.

Antigen binding proteins of the invention include partially human andfully human monoclonal antibodies that inhibit a biological activity ofIGF-1 and also inhibit a biological activity of IGF-2. One embodiment isdirected to a human monoclonal antibody that at least partially blocksbinding of IGF-1 and of IGF-2 to a cell that expresses human IGF-1R. Inone embodiment, the antibodies are generated by immunizing a transgenicmouse with an IGF-1R immunogen. In another embodiment, the immunogen isa human IGF-1R polypeptide (e.g., a soluble fragment comprising all orpart of the IGF-1R extracellular domain). Hybridoma cell lines derivedfrom such immunized mice, wherein the hybridoma secretes a monoclonalantibody that binds IGF-1R, also are provided herein.

Although human, partially human, or humanized antibodies will besuitable for many applications, particularly those involvingadministration of the antibody to a human subject, other types ofantigen binding proteins will be suitable for certain applications. Thenon-human antibodies of the invention can be, for example, derived fromany antibody-producing animal, such as mouse, rat, rabbit, goat, donkey,or non-human primate (such as monkey (e.g., cynomologous or rhesusmonkey) or ape (e.g., chimpanzee)). Non-human antibodies of theinvention can be used, for example, in in vitro and cell-culture basedapplications, or any other application where an immune response to theantibody of the invention does not occur, is insignificant, can beprevented, is not a concern, or is desired. In one embodiment, anon-human antibody of the invention is administered to a non-humansubject. In another embodiment, the non-human antibody does not elicitan immune response in the non-human subject. In another embodiment, thenon-human antibody is from the same species as the non-human subject,e.g., a mouse antibody of the invention is administered to a mouse. Anantibody from a particular species can be made by, for example,immunizing an animal of that species with the desired immunogen (e.g., asoluble IGF-1R polypeptide) or using an artificial system for generatingantibodies of that species (e.g., a bacterial or phage display-basedsystem for generating antibodies of a particular species), or byconverting an antibody from one species into an antibody from anotherspecies by replacing, e.g., the constant region of the antibody with aconstant region from the other species, or by replacing one or moreamino acid residues of the antibody so that it more closely resemblesthe sequence of an antibody from the other species. In one embodiment,the antibody is a chimeric antibody comprising amino acid sequencesderived from antibodies from two or more different species.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from theAfrican green monkey kidney cell line CVI (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, New York, 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) ofIGF-1R bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-IGF-1R antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-IGF-1R antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

In one aspect, the present invention provides antigen-binding fragmentsof an anti-IGF-1R antibody of the invention. Such fragments can consistentirely of antibody-derived sequences or can comprise additionalsequences. Examples of antigen-binding fragments include Fab, F(ab′)2,single chain antibodies, diabodies, triabodies, tetrabodies, and domainantibodies. Other examples are provided in Lunde et al., 2002, Biochem.Soc. Trans. 30:500-06.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol Biol. 178:379-87. Single chain antibodies derivedfrom antibodies provided herein include, but are not limited to, scFvscomprising the variable domain combinations L1H1, L2H2, L3H3, L4H4,L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14,L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23,L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31, L32H32,L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41,L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50,L51H51, and L52H52) are encompassed by the present invention.

Antigen binding proteins (e.g., antibodies, antibody fragments, andantibody derivatives) of the invention can comprise any constant regionknown in the art. The light chain constant region can be, for example, akappa- or lambda-type light chain constant region, e.g., a human kappa-or lambda-type light chain constant region. The heavy chain constantregion can be, for example, an alpha-, delta-, epsilon-, gamma-, ormu-type heavy chain constant regions, e.g., a human alpha-, delta-,epsilon-, gamma-, or mu-type heavy chain constant region. In oneembodiment, the light or heavy chain constant region is a fragment,derivative, variant, or mutein of a naturally occurring constant region.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.

In one embodiment, an antigen binding protein of the invention comprisesthe IgG1 heavy chain domain of FIG. 13 or a fragment of the IgG1 heavychain domain of FIG. 13. In another embodiment, an antigen bindingprotein of the invention comprises the kappa light chain constant chainregion of FIG. 13 or a fragment of the kappa light chain constant regionof FIG. 13. In another embodiment, an antigen binding protein of theinvention comprises both the IgG1 heavy chain domain, or a fragmentthereof, of FIG. 13 and the kappa light chain domain, or a fragmentthereof, of FIG. 13.

Accordingly, the antigen binding proteins of the present inventioninclude those comprising, for example, the variable domain combinationsL1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11,L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20,L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29,L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38,L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47,L48H48, L49H49, L50H50, L51H51, and L52H52, having a desired isotype(for example, IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD) as well asFab or F(ab′)₂ fragments thereof. Moreover, if an IgG4 is desired, itmay also be desired to introduce a point mutation (CPSCP→CPPCP) in thehinge region as described in Bloom et al., 1997, Protein Science 6:407,incorporated by reference herein) to alleviate a tendency to formintra-H chain disulfide bonds that can lead to heterogeneity in the IgG4antibodies.

Moreover, techniques for deriving antigen binding proteins havingdifferent properties (i.e., varying affinities for the antigen to whichthey bind) are also known. One such technique, referred to as chainshuffling, involves displaying immunoglobulin variable domain generepertoires on the surface of filamentous bacteriophage, often referredto as phage display. Chain shuffling has been used to prepare highaffinity antibodies to the hapten 2-phenyloxazol-5-one, as described byMarks et al., 1992, BioTechnology, 10:779.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for IGF-1R of at least 10⁶,measured as described in the Examples. In other embodiments, the antigenbinding proteins exhibit a K_(a) of at least 10⁷, at least 10⁸, at least10⁹, or at least 10¹⁰.

In another embodiment, the present invention provides an antigen bindingprotein that has a low dissociation rate from IGF-1R. In one embodiment,the antigen binding protein has a K_(off) of 1×10⁻⁴ s⁻¹ or lower. Inanother embodiment, the K_(off) is 5×10⁻⁵ s⁻¹ or lower. In anotherembodiment, the K_(off) is substantially the same as an antibody havinga combination of light chain and heavy chain variable domain sequencesselected from the group of combinations consisting of L1H1, L2H2, L3H3,L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13,L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22,L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31,L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40,L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49,L50H50, L51H51, and L52H52. In another embodiment, the antigen bindingprotein binds to IGF-1R with substantially the same K_(off) as anantibody that comprises one or more CDRs from an antibody having acombination of light chain and heavy chain variable domain sequencesselected from the group of combinations consisting of L1H1, L2H2, L3H3,L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13,L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22,L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31,L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40,L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49,L50H50, L51H51, and L52H52. In another embodiment, the antigen bindingprotein binds to IGF-1R with substantially the same K_(off) as anantibody that comprises one of the amino acid sequences illustrated inFIGS. 2 through 9. In another embodiment, the antigen binding proteinbinds to IGF-1R with substantially the same K_(off) as an antibody thatcomprises one or more CDRs from an antibody that comprises one of theamino acid sequences illustrated in FIGS. 2 through 9.

In another aspect, the present invention provides an antigen bindingprotein that binds to the L2 domain of human IGF-1R. Antigen bindingproteins that bind to the L2 domain can be made using any techniqueknown in the art. For example, such antigen binding proteins can beisolated using the full-length IGF-1R polypeptide (e.g., in amembrane-bound preparation), a soluble extracellular domain fragment ofIGF-1R (an example of which is provided in Example 1), or a smallerfragment of the IGF-1R extracellular domain comprising or consisting ofthe L2 domain (examples of which are provided in Example 10). Antigenbinding proteins so isolated can be screened to determine their bindingspecificity using any method known in the art (an example of which isprovided in Example 10).

In another aspect, the present invention provides an antigen bindingprotein that binds to human IGF-1R expressed on the surface of a celland, when so bound, inhibits IGF-1R signaling activity in the cellwithout causing a significant reduction in the amount of IGF-1R on thesurface of the cell. Any method for determining or estimating the amountof IGF-1R on the surface and/or in the interior of the cell can be used.In one embodiment, the present invention provides an antigen bindingprotein that binds to the L2 domain of a human IGF-1R expressed on thesurface of a cell and, when so bound, inhibits IGF-1R signaling activityin the cell without significantly increasing the rate of internalizationof the IGF-1R from the surface of the cell. In other embodiments,binding of the antigen binding protein to the IGF-1R-expressing cellcauses less than about 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, or0.1% of the cell-surface IGF-1R to be internalized. In another aspect,binding of the antigen binding protein to the IGF-1R-expressing cellcauses a gradual reduction in the amount of IGF-1R on the cell surfacesuch that within a few hours of contacting the cell with the antigenbinding protein, little or no decrease in cell surface IGF-1R isdetected, but, after several days or weeks of exposure of the cell tothe antigen binding protein, a marked decrease in cell surface IGF-1R isdetected.

In another aspect, the present invention provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO 00/09560, published Feb. 24, 2000, incorporated by reference.

The present invention further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of IGF-1R, or to anepitope of IGF-1R and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise an IGF-1R binding site from oneof the herein-described antibodies and a second IGF-1R binding regionfrom another of the herein-described antibodies, including thosedescribed herein by reference to other publications. Alternatively, abispecific antigen binding protein may comprise an antigen binding sitefrom one of the herein described antibodies and a second antigen bindingsite from another IGF-1R antibody that is known in the art, or from anantibody that is prepared by known methods or the methods describedherein.

Numerous methods of preparing bispecific antibodies are known in theart, and discussed in U.S. patent application Ser. No. 09/839,632, filedApr. 20, 2001 (incorporated by reference herein). Such methods includethe use of hybrid-hybridomas as described by Milstein et al., 1983,Nature 305:537, and others (U.S. Pat. No. 4,474,893, U.S. Pat. No.6,106,833), and chemical coupling of antibody fragments (Brennan et al.,1985, Science 229:81; Glennie et al., 1987, J. Immunol. 139:2367; U.S.Pat. No. 6,010,902). Moreover, bispecific antibodies can be produced viarecombinant means, for example by using leucine zipper moieties (i. e.,from the Fos and Jun proteins, which preferentially form heterodimers;Kostelny et al., 1992, J. Immnol. 148:1547) or other lock and keyinteractive domain structures as described in U.S. Pat. No. 5,582,996.Additional useful techniques include those described in Kortt et al.,1997, supra; U.S. Pat. No. 5,959,083; and U.S. Pat. No. 5,807,706.

In another aspect, the antigen binding protein of the present inventioncomprises a derivative of an antibody. The derivatized antibody cancomprise any molecule or substance that imparts a desired property tothe antibody, such as increased half-life in a particular use. Thederivatized antibody can comprise, for example, a detectable (orlabeling) moiety (e.g., a radioactive, colorimetric, antigenic orenzymatic molecule, a detecable bead (such as a magnetic or electrodense(e.g., gold) bead), or a molecule that binds to another molecule (e.g.,biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., aradioactive, cytotoxic, or pharmaceutically active moiety), or amolecule that increases the suitability of the antibody for a particularuse (e.g., administration to a subject, such as a human subject, orother in vivo or in vitro uses). Examples of molecules that can be usedto derivatize an antibody include albumin (e.g., human serum albumin)and polyethylene glycol (PEG). Albumin-linked and PEGylated derivativesof antibodies can be prepared using techniques well known in the art. Inone embodiment, the antibody is conjugated or otherwise linked totransthyretin (TTR) or a TTR variant. The TTR or TTR variant can bechemically modified with, for example, a chemical selected from thegroup consisting of dextran, poly(n-vinyl pyurrolidone), polyethyleneglycols, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols. USPat. App. No. 20030195154.

In another aspect, the present invention provides methods of screeningfor a molecule that binds to IGF-1R using the antigen binding proteinsof the present invention. Any suitable screening technique can be used.In one embodiment, an IGF-1R molecule, or a fragment thereof to which anantigen binding protein of the present invention binds, is contactedwith the antigen binding protein of the invention and with anothermolecule, wherein the other molecule binds to IGF-1R if it reduces thebinding of the antigen binding protein to IGF-1R. Binding of the antigenbinding protein can be detected using any suitable method, e.g., anELISA. Detection of binding of the antigen binding protein to IGF-1R canbe simplified by detectably labeling the antigen binding protein, asdiscussed above. In another embodiment, the IGF-1R-binding molecule isfurther analyzed to determine whether it inhibits IGF-1R, IGF-1, and/orIGF-2-mediated signaling.

Nucleic Acids

In one aspect, the present invention provides isolated nucleic acidmolecules. The nucleic acids comprise, for example, polynucleotides thatencode all or part of an antigen binding protein, for example, one orboth chains of an antibody of the invention, or a fragment, derivative,mutein, or variant thereof, polynucleotides sufficient for use ashybridization probes, PCR primers or sequencing primers for identifying,analyzing, mutating or amplifying a polynucleotide encoding apolypeptide, anti-sense nucleic acids for inhibiting expression of apolynucleotide, and complementary sequences of the foregoing. Thenucleic acids can be any length. They can be, for example, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides inlength, and/or can comprise one or more additional sequences, forexample, regulatory sequences, and/or be part of a larger nucleic acid,for example, a vector. The nucleic acids can be single-stranded ordouble-stranded and can comprise RNA and/or DNA nucleotides, andartificial variants thereof (e.g., peptide nucleic acids).

Nucleic acids encoding antibody polypeptides (e.g., heavy or lightchain, variable domain only, or full length) may be isolated fromB-cells of mice that have been immunized with IGF-1R. The nucleic acidmay be isolated by conventional procedures such as polymerase chainreaction (PCR).

FIG. 1 provides nucleic acid sequences encoding the variable regions ofthe heavy and light chain variable regions shown in FIGS. 2 and 3. Theskilled artisan will appreciate that, due to the degeneracy of thegenetic code, each of the polypeptide sequences in FIGS. 2 through 9also is encoded by a large number of other nucleic acid sequences. Thepresent invention provides each degenerate nucleotide sequence encodingeach antigen binding protein of the invention.

The invention further provides nucleic acids that hybridize to othernucleic acids (e.g., nucleic acids comprising a nucleotide sequence ofFIG. 1) under particular hybridization conditions. Methods forhybridizing nucleic acids are well-known in the art. See, e.g., CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. As defined herein, a moderately stringent hybridizationcondition uses a prewashing solution containing 5× sodiumchloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6×SSC, and a hybridizationtemperature of 55° C. (or other similar hybridization solutions, such asone containing about 50% formamide, with a hybridization temperature of42° C.), and washing conditions of 60° C., in 0.5×SSC, 0.1% SDS. Astringent hybridization condition hybridizes in 6×SSC at 45° C.,followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C.Furthermore, one of skill in the art can manipulate the hybridizationand/or washing conditions to increase or decrease the stringency ofhybridization such that nucleic acids comprising nucleotide sequencesthat are at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% identical toeach other typically remain hybridized to each other. The basicparameters affecting the choice of hybridization conditions and guidancefor devising suitable conditions are set forth by, for example,Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,chapters 9 and 11; and Current Protocols in Molecular Biology, 1995,Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4), and can be readily determined by those having ordinary skillin the art based on, for example, the length and/or base composition ofthe DNA.

Changes can be introduced by mutation into a nucleic acid, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anantigen binding protein) that it encodes. Mutations can be introducedusing any technique known in the art. In one embodiment, one or moreparticular amino acid residues are changed using, for example, asite-directed mutagenesis protocol. In another embodiment, one or morerandomly selected residues is changed using, for example, a randommutagenesis protocol. However it is made, a mutant polypeptide can beexpressed and screened for a desired property (e.g., binding to IGF-1Ror blocking the binding of IGF-1 and/or IGF-2 to IGF-1R).

Mutations can be introduced into a nucleic acid without significantlyaltering the biological activity of a polypeptide that it encodes. Forexample, one can make nucleotide substitutions leading to amino acidsubstitutions at non-essential amino acid residues. In one embodiment, anucleotide sequence provided in FIG. 1, or a desired fragment, variant,or derivative thereof, is mutated such that it encodes an amino acidsequence comprising one or more deletions or substitutions of amino acidresidues that are shown in FIGS. 2 through 9 to be residues where two ormore sequences differ. In another embodiment, the mutagenesis inserts anamino acid adjacent to one or more amino acid residues shown in FIGS. 2through 9 to be residues where two or more sequences differ.Alternatively, one or more mutations can be introduced into a nucleicacid that selectively change the biological activity (e.g., binding ofIGF-1R, inhibiting IGF-1 and/or IGF-2, etc.) of a polypeptide that itencodes. For example, the mutation can quantitatively or qualitativelychange the biological activity. Examples of quantitative changes includeincreasing, reducing or eliminating the activity. Examples ofqualitative changes include changing the antigen specificity of anantigen binding protein.

In another aspect, the present invention provides nucleic acid moleculesthat are suitable for use as primers or hybridization probes for thedetection of nucleic acid sequences of the invention. A nucleic acidmolecule of the invention can comprise only a portion of a nucleic acidsequence encoding a full-length polypeptide of the invention, forexample, a fragment that can be used as a probe or primer or a fragmentencoding an active portion (e.g., an IGF-1R binding portion) of apolypeptide of the invention.

Probes based on the sequence of a nucleic acid of the invention can beused to detect the nucleic acid or similar nucleic acids, for example,transcripts encoding a polypeptide of the invention. The probe cancomprise a label group, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used to identify acell that expresses the polypeptide.

In another aspect, the present invention provides vectors comprising anucleic acid encoding a polypeptide of the invention or a portionthereof. Examples of vectors include, but are not limited to, plasmids,viral vectors, non-episomal mammalian vectors and expression vectors,for example, recombinant expression vectors.

The recombinant expression vectors of the invention can comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell. The recombinant expression vectors includeone or more regulatory sequences, selected on the basis of the hostcells to be used for expression, which is operably linked to the nucleicacid sequence to be expressed. Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoterand cytomegalovirus promoter), those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences, see Voss et al., 1986, Trends Biochem. Sci.11:287, Maniatis et al., 1987, Science 236:1237, incorporated byreference herein in their entireties), and those that direct inducibleexpression of a nucleotide sequence in response to particular treatmentor condition (e.g., the metallothionin promoter in mammalian cells andthe tet-responsive and/or streptomycin responsive promoter in bothprokaryotic and eukaryotic systems (see id.). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

In another aspect, the present invention provides host cells into whicha recombinant expression vector of the invention has been introduced. Ahost cell can be any prokaryotic cell (for example, E. coli) oreukaryotic cell (for example, yeast, insect, or mammalian cells (e.g.,CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryoticcells via conventional transformation or transfection techniques. Forstable transfection of mammalian cells, it is known that, depending uponthe expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die), among other methods.

Indications

In one aspect, the present invention provides methods of treating asubject. The method can, for example, have a generally salubrious effecton the subject, e.g., it can increase the subject's expected longevity.Alternatively, the method can, for example, treat, prevent, cure,relieve, or ameliorate (“treat”) a disease, disorder, condition, orillness (“a condition”). Among the conditions to be treated inaccordance with the present invention are conditions characterized byinappropriate expression or activity of IGF-1, IGF-2, and/or IGF-1R. Insome such conditions, the expression or activity level is too high, andthe treatment comprises administering an IGF-1R antagonist as describedherein. In other such conditions, the expression or activity level istoo low, and the treatment comprises administering an IGF-1R agonist asdescribed herein.

One example of a type of condition that can be treated using the methodsand compositions of the present invention is a condition that involvescell growth, for example, a cancerous condition. Thus, in oneembodiment, the present invention provides compositions and methods fortreating a cancerous condition. The cancerous condition can be anycancerous condition that can be treated using the compositions comprisedherein, for example, IGF-1R antagonizing antigen binding proteins suchas anti-IGF-1R antibodies, antibody fragments, or antibody derivatives.Examples of cancerous conditions include, for example, AcuteLymphoblastic Leukemia, Adrenocortical Carcinoma, AIDS-Related Cancers,AIDS-Related Lymphoma, Anal Cancer, Childhood Cerebellar Astrocytoma,Childhood Cerebral Astrocytoma, Basal Cell Carcinoma, Extrahepatic BileDuct Cancer, Bladder Cancer, Osteosarcoma/Malignant Fibrous HistiocytomaBone Cancer, Brain Tumors (e.g., Brain Stem Glioma, CerebellarAstrocytoma, Cerebral Astrocytoma/Malignant Glioma, Ependymoma,Medulloblastoma, Supratentorial Primitive Neuroectodermal Tumors, VisualPathway and Hypothalamic Glioma), Breast Cancer, BronchialAdenomas/Carcinoids, Burkitt's Lymphoma, Carcinoid Tumor,Gastrointestinal Carcinoid Tumor, Carcinoma of Unknown Primary, PrimaryCentral Nervous System, Cerebellar Astrocytoma, CerebralAstrocytoma/Malignant Glioma, Cervical Cancer, Childhood Cancers,Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, ChronicMyeloproliferative Disorders, Colon Cancer, Colorectal Cancer, CutaneousT-Cell Lymphoma, Endometrial Cancer, Ependymoma, Esophageal Cancer,Ewing's Family of Tumors, Extracranial Germ Cell Tumor, ExtragonadalGerm Cell Tumor, Extrahepatic Bile Duct Cancer, Intraocular Melanoma EyeCancer, Retinoblastoma Eye Cancer, Gallbladder Cancer, Gastric (Stomach)Cancer, Gastrointestinal Carcinoid Tumor, Germ Cell Tumors (e.g.,Extracranial, Extragonadal, and Ovarian), Gestational TrophoblasticTumor, Glioma (e.g., Adult, Childhood Brain Stem, Childhood CerebralAstrocytoma, Childhood Visual Pathway and Hypothalamic), Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin'sLymphoma, Hypopharyngeal Cancer, Hypothalamic and Visual Pathway Glioma,Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas),Kaposi's Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal Cancer, Leukemia(e.g., Acute Lymphoblastic, Acute Myeloid, Chronic Lymphocytic, ChronicMyelogenous, and Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer,Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Lymphoma (e.g.,AIDS-Related, Burkitt's, Cutaneous T-Cell, Hodgkin's, Non-Hodgkin's, andPrimary Central Nervous System), Waldenstrom's Macroglobulinemia,Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, Medulloblastoma,Melanoma, Intraocular (Eye) Melanoma, Merkel Cell Carcinoma,Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary,Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma CellNeoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Diseases, Myelogenous Leukemia,Chronic Myeloid Leukemia, Multiple Myeloma, Chronic MyeloproliferativeDisorders, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Oral Cancer, Oropharyngeal Cancer,Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Islet Cell PancreaticCancer, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer,Penile Cancer, Pheochromocytoma, Pineoblastoma, Pituitary Tumor, PlasmaCell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, PrimaryCentral Nervous System Lymphoma, Prostate Cancer, Rectal Cancer, RenalCell (Kidney) Cancer, Renal Pelvis and Ureter Transitional Cell Cancer,Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Soft TissueSarcoma, Uterine Sarcoma, Sezary Syndrome, non-Melanoma Skin Cancer,Merkel Cell Skin Carcinoma, Small Intestine Cancer, Soft Tissue Sarcoma,Squamous Cell Carcinoma, Cutaneous T-Cell Lymphoma, Testicular Cancer,Thymoma, Thymic Carcinoma, Thyroid Cancer, Gestational TrophoblasticTumor, Carcinoma of Unknown Primary Site, Cancer of Unknown PrimarySite, Urethral Cancer, Endometrial Uterine Cancer, Uterine Sarcoma,Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer,Waldenstrom's Macroglobulinemia, and Wilms' Tumor.

Four different groups have studied a total of 425 breast cancers, mostlyductal in origin, and 48 normal tissues or benign specimens byradioimmunoassay (“RIA”) or immunohistochemistry (“IHC”) (Papa et al.,1993, Cancer Research 53: 3736-40, Happerfield et al., 1997, Journal ofPathology 183: 412-17; Ellis et al., 1998, Breast Cancer Research &Treatment 52: 175-84, Lee et al., 1998, Breast Cancer Research &Treatment 47: 295-302, Schnarr et al., 2000, International Journal ofCancer 89: 506-13). These studies suggest that elevated IGF-1Rexpression, on the order of 5-10 fold, is associated with favorableprognosis and biomarkers (ER+PR+), suggesting that estrogen and IGFcooperate in the maintenance or progression of well differentiatedtumor. Similarly, estrogen has been shown to be essential for the growthand survival of the ER+MCF-7 breast cancer cell line, and in thiscontext IGF-1R is up-regulated by estrogen treatment (reviewed in Elliset al., 1998, Breast Cancer Research & Treatment 52: 175-84). Thus, inone embodiment, the present invention provides a method of treatingbreast cancer in a subject in need of such treatment, comprisingadministering to the subject an effective amount of an IGF-1R antagonistas described herein. In another embodiment, the method further comprisesadministering a hormone inhibitor, e.g., an estrogen inhibitor.

A retrospective IGF-1R IHC analysis has been reported for a collectionof 12 colonic adenomas, 36 primary colorectal adenocarcinomas and 27corresponding metastases, and 34 adjacent normal tissues (Hakam et al.,1999, Human Pathology. 30: 1128-33). The frequency of moderate to strongIHC staining appeared to dramatically increase with higher stage andtumor grade (0% normal vs. 93% metastases). The results are consistentwith RNA analysis by RNAse protection assay (“RPA”) (Freier et al.,1999, Gut 44: 704-08). Thus, in one embodiment, the present inventionprovides a method of treating colon cancer in a subject in need of suchtreatment, comprising administering to the subject an effective amountof an IGF-1R antagonist as described herein.

High plasma IGF-1 and reduced IGFbp3 in men 40-80 years old isassociated with increased prostate cancer risk (Chan et al., 1998,Science 279: 563-6). High IGF-1 is associated with a risk of othercancers including breast (Hankinson et al., 1998, Lancet 351: 1393-96),colon (Ma et al., 1999, Journal of the National Cancer Institute 91:620-25) and lung (Yu et al., 1999, Journal of the National CancerInstitute 91: 151-56). In transgenic mouse models, tumor incidence isincreased by IGF-1 overexpression in diverse locations (Bol et al.,1997, Oncogene 14: 1725-34; DiGiovanni et al., 2000, Cancer Research 60:1561-70; DiGiovanni et al., 2000, Proceedings of the National Academy ofSciences of the United States of America 97: 3455-60, Hadsell et al.,2000, Oncogene 19: 889-98). These mouse studies point to a role for bothserum and stromal produced IGF-1. Thus, in one embodiment, the presentinvention provides a method of treating a subject in need of suchtreatment, comprising administering to the subject an effective amountof an antagonist of IGF-1R as described herein, wherein the antagonistinhibits the activation of IGF-1R by IGF-1. In another embodiment, thesubject has cancer. In another embodiment, the subject has a tumor. Inanother embodiment, the cancer is prostate, breast, colon or lungcancer.

It has been observed that bone is the major source of IGF-1 in the body.Thus, in one aspect, the present invention provides compositions andmethods for inhibiting IGF-1R in a bone of a subject. In one embodiment,an IGF-1R inhibitor of the present invention is administered to asubject that has, or is at risk for developing, a tumor in a bone. Thetumor can be, for example, a primary tumor or a metastatic tumor. Thetreatment optionally further comprises administering to the subject oneor more additional therapeutic and/or palliative treatments, forexample, an anti-tumor treatment (e.g., chemotherapy, radiation therapy,or anti-hormone therapy) or a treatment that inhibits bone turnover(e.g., denosumab (Amgen Inc., Thousand Oaks, Calif.)).

IGF-2 is overexpressed in a variety of tumors and stromal tissues. IGF-2levels appear especially high (as much as 40 fold) in primary livercancers (Cariani et al., 1988, Cancer Research 48: 6844-49) andadenocarcinoma of the colon (Freier et al., 1999, Gut 44: 704-08). Manyof the overgrowth disorders are associated with an increased incidenceof childhood tumors. Five to ten percent of individuals with either theprenatal growth disorder Beckwith-Weidmann Syndrome (BWS) orhemihyperplasia develop tumors such as nephroblastoma, adrenalcarcinoma, and neuroblastoma (reviewed by Morison et al., 1998,Molecular Medicine Today 4: 110-05). The tumor-predisposing factor inthese children appears to be the mosaic loss of maternal IGF-2 geneimprinting, or duplication of the paternal chromosomal arm (11p) thatcarries IGF-2. Both alterations would increase the level of IGF-2expression. IGF-2 overexpression as a result of mosaic uniparentaldisomy or loss of IGF-2 imprinting has also been detected in Wilmstumors. Growth disorders are not observed in these children even thoughthe IGF-2 gene alterations also occur in some normal tissues, perhapsreflecting the tissue distribution of the affected cells. Imprinting ofthe maternal IGF-2 gene also occurs in mice, and the effects of IGF-2overexpression are consistent with the human situation (Cariani et al.,1991, Journal of Hepatology 13: 220-26, Schirmacher et al., 1992, CancerResearch 52: 2549-56; Harris et al., 1998, Oncogene 16: 203-09). Theincidence of tumors and organomegaly increases in mice thattransgenically express excess IGF-2 (Christofori et al., 1994, Nature369: 414-18, Ward et al., 1994, Proceedings of the National Academy ofSciences of the United States of America 91: 10365-9, Wolf et al., 1994,Endocrinology 135: 1877-86, Bates et al., 1995, British Journal ofCancer 72: 1189-93, Hassan et al., 2000, Cancer Research 60: 1070-76).Local IGF-2 overexpression increases the spontaneous appearance ofprostate, mammary, intestinal, liver and epidermal tumors. Plasmaspecific expression using liver promoters elevate hepatocellularcarcinomas and lymphoma. Thus, in one embodiment, the present inventionprovides a method of treating a subject in need of such treatment,comprising administering to the subject an effective amount of anantagonist of IGF-1R as described herein, wherein the antagonistinhibits the activation of IGF-1R by IGF-2. In another embodiment, thesubject has cancer. In another embodiment, the subject has a tumor. Inanother embodiment, the subject has liver cancer, adenocarcinoma of thecolon, Beckwith-Weidmann Syndrome, hemihyperplasia, nephroblastoma,adrenal carcinoma, neuroblastoma, mosaic loss of maternal IGF-2 geneimprinting, duplication of the paternal chromosomal arm (11p), increasedIGF-2 expression, a tumor (e.g., a prostate, mammary, intestinal, liver,epidermal, or Wilms tumor), organomegaly, hepatocellular carcinoma, orlymphoma.

In another aspect, the invention provides methods of preventing orinhibiting a cancer from spreading to another part of the body, or oftreating a cancer that has spread to another part of the body. In oneembodiment, the cancer has spread to a regional lymph node. In anotherembodiment, the cancer is metastatic. The primary tumor can be any kindof tumor, for example, an adenocarcinoma tumor (e.g., a prostateadenocarcinoma tumor, a breast carcinoma tumor, or a renal cellcarcinoma tumor), a non-small cell or small cell lung cancer tumor, athyroid cancer tumor, etc. The site of the metastatic tumor can beanywhere in the body. It can be, for example, in bone, the lymph system,lung, brain, eye, skin, pancrease, or liver. In one particularembodiment, a subject having a tumor disease is treated with aneffective amount of an IGF-1R inhibiting composition of the presentinvention such that the primary tumor is prevented from metastasizing.In another particular embodiment, a subject having a primary tumor istreated with an effective amount of an IGF-1R inhibiting composition ofthe present invention such that the primary tumor is inhibited frommetastasizing. In another particular embodiment, a subject having ametastatic tumor is treated with an effective amount of an IGF-1Rinhibiting composition of the present invention such that growth orspreading of the secondary tumor is inhibited. In another particularembodiment, a subject having a metastatic tumor is treated with aneffective amount of an IGF-1R inhibiting composition of the presentinvention such that the secondary tumor is reduced in size. In a moreparticular embodiment, the primary tumor is an adenocarcinoma tumor, anon-small cell lung tumor, a small cell lung tumor, or a thyroid cancer.In another more particular embodiment, the metastatic tumor is in abone. In another more particular embodiment, a metastatic tumor isprevented or inhibited from forming in a bone. In another moreparticularly defined embodiment, the method comprises treating thesubject with an IGF-1R inhibiting composition of the present inventionand one or more other treatments (e.g., a treatment that kills orinhibits the growth of cancer cells, such as radiation, hormonaltherapy, or chemotherapy, or a treatment that inhibits the turnover ofbone, such as denosumab), non-limiting examples of which are providedherein. The one or more other treatments can include, for example thestandard of care for the subject's particular condition and/orpalliative care.

Without being bound to any particular theory, tumor cells appear todepend on the PI3 Kinase/Akt signaling pathway to resist theapoptosis-inducing activity of chemotherapeutics, radiation, andanti-hormone therapy. Thus, in one embodiment, the present inventionprovides methods of treating a subject in need of such treatmentcomprising administering to the subject an IGF-1R antagonist of thepresent invention and a chemotherapeutic, radiation, and/or ananti-hormone therapy. This concept has been validated experimentally incell culture models and rodent tumor models by antisense and dominantnegative mutations (reviewed by Baserga et al., 1997, Biochimica etBiophysica Acta 1332: F105-26, Baserga, 2000, Oncogene 19: 5574-81). Inone embodiment, the chemotherapeutic agents is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, anti-survival agents,biological response modifiers, anti-hormones, e.g. anti-androgens, andanti-angiogenesis agents.

One example of a chemotherapeutic agent that can be administered incombination with an IGF-1 receptor inhibitor of the invention is CPT-11.CPT-11 (Irinotecan hydorchloride trihydrate) is a semi synthetic, watersoluble derivative of camptothecin, a plant alkaloid. CPT-11 and anassociated metabolite called SN38 inhibit topoisomerase 1 (TOPO1). Thisenzyme introduces reversible single-strand breaks in DNA that allowunwinding and permit DNA replication to proceed. Inhibition of TOPO1prevents religation of single-strand breaks after DNA replicationresulting in greatly increased chromosomal fragmentation. This DNAdamage promotes cell death by apoptosis through the action of p53 andother systems that monitor genome integrity. The cytotoxic effect ofCPT-11 is generally limited to cells that are replicating DNA (S-Phase).Quiescent cells are largely unaffected.

In another embodiment, the present invention provides treating a subjectin need thereof with an effective amount of an IGF-1R antagonist of thepresent invention and with an effective amount of an apoptosis-inducingagent.

In another embodiment, an anti-angiogenesis agent, such as an MMP-2(matrix-metalloproteinase 2) inhibitor, an MMP-9(matrix-metalloproteinase 9) inhibitor, and/or a COX-II (cyclooxygenaseII) inhibitor, is used in conjunction with a compound of the invention.Examples of useful COX-II inhibitors include CELEBREX™ (alecoxib),BEXTRA™ (valdecoxib), and VIOXX™ (rofecoxib). Examples of useful matrixmetalloproteinase inhibitors are described in WO 96/33172 (publishedOct. 24, 1996), WO 96/27583 (published Mar. 7, 1996), European PatentApplication No. 97304971.1 (filed Jul. 8, 1997), European PatentApplication No. 99308617.2 (filed Oct. 29, 1999), WO 98/07697 (publishedFeb. 26, 1998), WO 98/03516 (published Jan. 29, 1998), WO 98/34918(published Aug. 13, 1998), WO 98/34915 (published Aug. 13, 1998), WO98/33768 (published Aug. 6, 1998), WO 98/30566 (published Jul. 16,1998), European Patent Publication 606,046 (published Jul. 13, 1994),European Patent Publication 931,788 (published Jul. 28, 1999), WO90/05719 (published May 31, 1990), WO 99/52910 (published Oct. 21,1999), WO 99/52889 (published Oct. 21, 1999), WO 99/29667 (publishedJun. 17, 1999), PCT International Application No. PCT/IB98/01113 (filedJul. 21, 1998), European Patent Application No. 99302232.1 (filed Mar.25, 1999), Great Britain patent application number 9912961.1 (filed Jun.3, 1999), U.S. Provisional Application No. 60/148,464 (filed Aug. 12,1999), U.S. Pat. No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No.5,861,510 (issued Jan. 19, 1999), and European Patent Publication780,386 (published Jun. 25, 1997), all of which are incorporated hereinin their entireties by reference. In one embodiment, the MMP inhibitoris one that does not demonstrate arthralgia. In another embodiment, theMMP inhibitor selectively inhibits MMP-2 and/or MMP-9 relative to othermatrix-metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specificexamples of MMP inhibitors useful in the present invention are AG-3340,RO 32-3555, RS 13-0830, and the compounds recited in the following list:3-[[4-(4-fluorophenoxy)-benzene-sulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluorophenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluorobenzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluorophenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluorophenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydropyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[4-(4-fluorophenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionicacid;3-[[4-(4-fluorophenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluorophenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluorophenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts, solvates,derivatives, and other preparations of the compounds.

Sporadic mutations that inactivate the PETN gene product occurrelatively frequently in most human cancers (Yamada et al., 2001, J CellSci 114:2375-82, Hill et al., 2002, Pharmacol Therapeut 93:243-51). Lossof PTEN causes the Akt phosphorylated state to persist through loss ofthe ability to down-regulate stimulatory signals originating from IGF-1Rand other sources. The status of the p53 tumor suppressor alsoinfluences the activity of the IGF-1R signaling system. In the groundstate, the basal or constitutive transcription of IGF-1R is repressed byp53 via an indirect mechanism. Activation of Akt promotes thephosphorylation of mdm2, which then binds the p53 tumor suppressor andpromotes its degradation (Mayo et al., 2002, TIBS 27:462-67), resultingin increased IGF-1R expression. A similar outcome is observed when p53is inactivated by mutation. When transiently expressed in Saos-2 (ahuman osteosarcoma cell line) and RD (a rhabdomyosarcoma cell line),wild-type p53 is able to suppress the activity of a cotransfected IGF-1Rpromoter construct, whereas tumor-derived, mutant versions of p53 haveno effect. It has been proposed that the increased level of IGF-1Rpromotes the resistance to apoptosis associated with p53 loss inmalignant cells (Werner et al., 2000, Cell Mol Life Sci 57:932-42).Thus, in one embodiment, the present invention provides a method oftreating a cancerous condition in a subject in need of such treatmentcomprising administering to the subject an effective amount of an IGF-1Rantagonist as described herein, wherein the cancerous condition ischaracterized by cells that have a reduced expression or activity ofp53.

The WT1 (Wilms kidney tumor suppressor 1 protein) also has been shown tobind and repress the IGF-1R promoter. Thus, in one embodiment, thepresent invention provides a method of treating a cancerous condition ina subject in need of such treatment comprising administering to thesubject an effective amount of an IGF-1R antagonist as described hereinwherein the cancerous condition is characterized by a reduced expressionor activity of WT 1.

The proliferation of normal fibroblasts has been shown to require, underdefined culture conditions, the combined action of IGF and a stromalgrowth factor (e.g. PDGF, EGF) to ramp-up Ras/Raf/Map Kinase and promotecell cycle entry (the G0 to G1 transition). Fibroblasts derived fromIGF-1R (−/−) mice do not respond to growth factor alone, or mostoncogenes (e.g. oncogenic Ras) that activate the Ras/Raf/Map Kinasepathway. Thus, in one embodiment, the present invention provides amethod of treating a subject in need of such treatment comprisingadministering to the subject an IGF-1R antagonist as described hereinand an agent that targets a growth factor and/or a growth factorreceptor, such as a growth factor receptor tyrosine kinase, e.g., theEGFR, HER-2, bcr-abl, VEGFR, Kit, raf, mTOR, CDK1/2, VEGFR2, PKCI3, Mek,and/or KDR. Examples of molecules that target such growth factors and/orreceptors include panitumumab (Abgenix, Fremont, Calif./Amgen, ThousandOaks, Calif.), HERCEPTIN™ (Genentech, South San Francisco, Calif.),GLEEVEC™ (Novartis, East Hanover, N.J.), IRESSA™ (AstraZeneca,Wilmington, Del.), ERBITUX™, (ImClone, New York, N.Y.), AVASTIN™,(Genentech), PTK787 (Novartis), SU11248 (Pfizer, New York, N.Y.),TARCEVA™ (OSI Pharmaceuticals, Melville, N.Y.), 43-9006 (Bayer, WestHaven, Conn.), CCI-779 (Wyeth, Madison, N.J.), RAD001 (Novartis),BMS-387032 (Bristol-Myers Squibb, New York, N.Y.), IMC-1C11 (ImClone),LY333531 (Eli Lilly, Indianapolis, Ind.), PD 184352 (Pfizer), 2C4(Genentech), and GW2016 (GlaxoSmithKline, Research Triangle Park, N.C.).

The role of IGF-1R in hematological malignancies has been reviewed by(Novak et al., 2003, Insulin-Like Growth Factors and HematologicalMalignancies in Insulin-Like Growth Factors, LeRoith et al., eds.,Landes Bioscience). A functional role for the IGF-1R in hematopoieticmalignancies is demonstrated by, for example, the ability of IGF-1Rmonoclonal antibodies to block transformed cell growth in culture. IGF-Ihas been found to enhance growth of freshly isolated human acutemyelogenous leukemia and acute lymphoblastic leukemia blasts. Withrespect to T cell malignancies, IGF-I has been shown to influence thegrowth of murine lymphoma cells bearing a pre-T cell phenotype and,immature and mature primary human T lineage acute lymphoblastic leukemiacells were found to express high numbers of IGF-1R. Thus, in oneembodiment, the present invention provides methods of treating ahematological malignancy in a subject in need thereof comprisingadministering to the subject an antagonist of IGF-1R as describedherein. In another embodiment, the malignancy is an acute myelogenousleukemia, an acute lymphoblastic leukemia, or a T cell malignancy.

In another aspect, the present invention provides methods of identifyingsubjects who are more likely to benefit from treatment using thecompositions and/or methods of treatment of the present invention. Suchmethods can enable a caregiver to better tailor a therapeutic regimen toa particular subject's needs and reduce the likelihood of an ineffectiveor counterproductive course of treatment. In one embodiment, the presentinvention provides a method of determining whether a subject is acandidate for treatment using a composition or method as describedherein comprising determining whether a target cell type in the subjectexpresses IGF-1R, wherein if the target cell type expresses IGF-1R, thenthe subject is a candidate for treatment. In another embodiment, themethod comprises determining the approximate average number of IGF-1Rmolecules per target cell, wherein 10², 10³, 10⁴, 10⁵, or 10⁶ IGF-1R percell indicates that the subject is a candidate for treatment. Theapproximate average number of IGF-1R molecules per target cell can bedetermined using any technique known in the art, for example, bystaining a sample comprising cells of the target cell type with anIGF-1R binding molecule, and detecting the amount of IGF-1R bindingmolecule bound to the sample, where the amount of IGF-1R bindingmolecule detected is proportional to the average number of IGF-1Rmolecules in the sample. In another embodiment, the method comprisescomparing the approximate average number of IGF-1R molecules per targetcell to a reference standard, wherein if the approximate average numberof IGF-1R molecules per target cell is greater than the referencestandard, then the subject is more likely to benefit from treatmentusing the compositions and/or methods of treatment of the presentinvention. In another embodiment, the target cell type is a cancerouscell type. In another embodiment, the target cell type is a colon cancercell type, a breast cancer cell type, an NSCLC cell type, or a leukemiccell type.

In another embodiment, a subject who is a candidate for treatment isidentified by detecting IGF-1 and/or IGF-2 in the target cell type, orin the stratum of the target cell type. In another embodiment, thetarget cell type is a cancerous cell type. In another embodiment, thetarget cell type is a colon cancer cell type, a breast cancer cell type,an NSCLC cell type, or a leukemic cell type.

In another embodiment, a subject who is a candidate for treatment isidentified by detecting activity of IGF-1R-mediated signaling in thetarget cell type, wherein IGF-1R-mediated signaling in the target celltype indicates that the subject is a candidate for treatment. Examplesof molecules that can be monitored for IGF-1R-dependent changes areshown in FIG. 10, such as molecules in the PI3/Akt pathway, e.g.,IGF-1R, IRS adapters, Akt, etc. Such molecules can be monitored for, forexample, a change in phosphorylation status, e.g., an increase inphosphorylation. Phosphospecific antibodies that recognize the activatedforms of these protein markers are highly developed, and these reagentshave proven to be reliable for immunoblot detection in experimentalsystems.

The compositions and/or methods of the present invention also can beused, for example, in cosmetic treatments, in veterinary treatments, toincrease longevity, to treat reproductive defects, and to treat avariety of growth related disorders.

Therapeutic Methods and Administration of Antigen Binding Proteins

Certain methods provided herein comprise administering an IGF-1R bindingantigen binding protein to a subject, thereby reducing an IGF-1-inducedbiological response that plays a role in a particular condition. Inparticular embodiments, methods of the invention involve contactingendogenous IGF-1R with an IGF-1R binding antigen binding protein, e.g.,via administration to a subject or in an ex vivo procedure.

The term “treatment” encompasses alleviation or prevention of at leastone symptom or other aspect of a disorder, or reduction of diseaseseverity, and the like. An antigen binding protein need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method comprisingadministering to a patient an IGF-1R antagonist in an amount and for atime sufficient to induce a sustained improvement over baseline of anindicator that reflects the severity of the particular disorder.

As is understood in the pertinent field, pharmaceutical compositionscomprising the molecules of the invention are administered to a subjectin a manner appropriate to the indication. Pharmaceutical compositionsmay be administered by any suitable technique, including but not limitedto parenterally, topically, or by inhalation. If injected, thepharmaceutical composition can be administered, for example, viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal or subcutaneous routes, by bolus injection, orcontinuous infusion. Localized administration, e.g. at a site of diseaseor injury is contemplated, as are transdermal delivery and sustainedrelease from implants. Delivery by inhalation includes, for example,nasal or oral inhalation, use of a nebulizer, inhalation of theantagonist in aerosol form, and the like. Other alternatives includeeyedrops; oral preparations including pills, syrups, lozenges or chewinggum; and topical preparations such as lotions, gels, sprays, andointments.

Use of antigen binding proteins in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an antigen binding protein that binds IGF-1R ex vivo.The antigen binding protein may be bound to a suitable insoluble matrixor solid support material.

Advantageously, antigen binding proteins are administered in the form ofa composition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents, for example, a second IGF-1 receptor-inhibitingsubstance, an anti-angiogenic substance, a chemotherapeutic substance,an analgesic substance, etc., non-exclusive examples of which areprovided herein. In various particular embodiments, the compositioncomprises one, two, three, four, five, or six physiologically activeagents in addition to an IGF-1R binding antigen binding protein

In one embodiment, the pharmaceutical composition comprise an antigenbinding protein of the invention together with one or more substancesselected from the group consisting of a buffer, an antioxidant such asascorbic acid, a low molecular weight polypeptide (such as those havingfewer than 10 amino acids), a protein, an amino acid, a carbohydratesuch as glucose, sucrose or dextrins, a chelating agent such as EDTA,glutathione, a stabilizer, and an excipient. Neutral buffered saline orsaline mixed with conspecific serum albumin are examples of appropriatediluents. In accordance with appropriate industry standards,preservatives such as benzyl alcohol may also be added. The compositionmay be formulated as a lyophilizate using appropriate excipientsolutions (e.g., sucrose) as diluents. Suitable components are nontoxicto recipients at the dosages and concentrations employed. Furtherexamples of components that may be employed in pharmaceuticalformulations are presented in Remington's Pharmaceutical Sciences,16^(th) Ed. (1980) and 20^(th) Ed. (2000), Mack Publishing Company,Easton, Pa.

Kits for use by medical practitioners include an IGF-1receptor-inhibiting substance of the invention and a label or otherinstructions for use in treating any of the conditions discussed herein.In one embodiment, the kit includes a sterile preparation of one or moreIGF-1R binding antigen binding proteins, which may be in the form of acomposition as disclosed above, and may be in one or more vials.

Dosages and the frequency of administration may vary according to suchfactors as the route of administration, the particular antigen bindingproteins employed, the nature and severity of the disease to be treated,whether the condition is acute or chronic, and the size and generalcondition of the subject. Appropriate dosages can be determined byprocedures known in the pertinent art, e.g. in clinical trials that mayinvolve dose escalation studies.

An IGF-1 receptor inhibiting substance of the invention may beadministered, for example, once or more than once, e.g., at regularintervals over a period of time. In particular embodiments, an antigenbinding protein is administered over a period of at least a month ormore, e.g., for one, two, or three months or even indefinitely. Fortreating chronic conditions, long-term treatment is generally mosteffective. However, for treating acute conditions, administration forshorter periods, e.g. from one to six weeks, may be sufficient. Ingeneral, the antigen binding protein is administered until the patientmanifests a medically relevant degree of improvement over baseline forthe chosen indicator or indicators.

Particular embodiments of the present invention involve administering anantigen binding protein at a dosage of from about 1 ng of antigenbinding protein per kg of subject's weight per day (“1 ng/kg/day”) toabout 10 mg/kg/day, more preferably from about 500 ng/kg/day to about 5mg/kg/day, and most preferably from about 5 μg/kg/day to about 2mg/kg/day, to a subject. In additional embodiments, an antigen bindingprotein is administered to adults one time per week, two times per week,or three or more times per week, to treat an IGF-1 and/or IGF-2 mediateddisease, condition or disorder, e.g., a medical disorder disclosedherein. If injected, the effective amount of antigen binding protein peradult dose may range from 1-20 mg/m², and preferably is about 5-12mg/m². Alternatively, a flat dose may be administered; the amount mayrange from 5-100 mg/dose. One range for a flat dose is about 20-30 mgper dose. In one embodiment of the invention, a flat dose of 25 mg/doseis repeatedly administered by injection. If a route of administrationother than injection is used, the dose is appropriately adjusted inaccordance with standard medical practices. One example of a therapeuticregimen involves injecting a dose of about 20-30 mg of antigen bindingprotein to one to three times per week over a period of at least threeweeks, though treatment for longer periods may be necessary to inducethe desired degree of improvement. For pediatric subjects (age 4-17),one exemplary suitable regimen involves the subcutaneous injection of0.4 mg/kg, up to a maximum dose of 25 mg of antigen binding proteinadministered two or three times per week.

Particular embodiments of the methods provided herein involvesubcutaneous injection of from 0.5 mg to 10 mg, preferably from 3 to 5mg, of an antigen binding protein, once or twice per week. Anotherembodiment is directed to pulmonary administration (e.g., by nebulizer)of 3 or more mg of antigen binding protein once a week.

Examples of therapeutic regimens provided herein comprise subcutaneousinjection of an antigen binding protein once a week, at a dose of 1.5 to3 mg, to treat a condition in which IGF-1R signaling plays a role.Examples of such conditions are provided herein and include, forexample, cancer, acromegaly and other overgrowth disorders, diabetes,obesity, macular degeneration, and aging. Weekly administration ofantigen binding protein is continued until a desired result is achieved,e.g., the subject's symptoms subside. Treatment may resume as needed,or, alternatively, maintenance doses may be administered.

Other examples of therapeutic regimens provided herein comprisesubcutaneous or intravenous administration of a dose of 1, 3, 5, 6, 7,8, 9, 10, 11, 12, 15, or 20 milligrams of an IGF-1R inhibitor of thepresent invention per kilogram body mass of the subject (mg/kg). Thedose can be administered once to the subject, or more than once at acertain interval, for example, once a day, three times a week, twice aweek, once a week, three times a month, twice a month, once a month,once every two months, once every three months, once every six months,or once a year. The duration of the treatment, and any changes to thedose and/or frequency of treatment, can be altered or varied during thecourse of treatment in order to meet the particular needs of thesubject.

In another embodiment, an antigen binding protein is administered to thesubject in an amount and for a time sufficient to induce an improvement,preferably a sustained improvement, in at least one indicator thatreflects the severity of the disorder that is being treated. Variousindicators that reflect the extent of the subject's illness, disease orcondition may be assessed for determining whether the amount and time ofthe treatment is sufficient. Such indicators include, for example,clinically recognized indicators of disease severity, symptoms, ormanifestations of the disorder in question. In one embodiment, animprovement is considered to be sustained if the subject exhibits theimprovement on at least two occasions separated by two to four weeks.The degree of improvement generally is determined by a physician, whomay make this determination based on signs, symptoms, biopsies, or othertest results, and who may also employ questionnaires that areadministered to the subject, such as quality-of-life questionnairesdeveloped for a given disease.

Elevated levels of IGF-1 and/or IGF-2 are associated with a number ofdisorders, including, for example, cancer (e.g., lung, prostate, breastand colon cancers), and acromegaly and other overgrowth disorders (e.g.,constitutionally tall children). Subjects with a given disorder may bescreened, to identify those individuals who have elevated IGF-1 and/orIGF-2 levels, thereby identifying the subjects who may benefit most fromtreatment with an IGF-1R binding antigen binding protein. Thus,treatment methods provided herein optionally comprise a first step ofmeasuring a subject's IGF-1 and/or IGF-2 levels. An antigen bindingprotein may be administered to a subject in whom IGF-1 and/or IGF-2levels are elevated above normal. In one embodiment, the presentinvention provides a method of treating an overgrowth disorder (e.g.,acromegaly) comprising administering to a subject in need thereof anantigen binding protein of the present invention and pegvisomant.

A subject's levels of IGF-1 and/or IGF-2 may be monitored before, duringand/or after treatment with an antigen binding protein, to detectchanges, if any, in their levels. For some disorders, the incidence ofelevated IGF-1 and/or IGF-2 levels may vary according to such factors asthe stage of the disease or the particular form of the disease. Knowntechniques may be employed for measuring IGF-1 and/or IGF-2 levels,e.g., in a subject's serum. IGF-1 and/or IGF-2 levels in blood samplesmay be measured using any suitable technique, for example, ELISA.

Particular embodiments of methods and compositions of the inventioninvolve the use of an antigen binding protein and one or more additionalIGF-1R antagonists, for example, two or more antigen binding proteins ofthe invention, or an antigen binding protein of the invention and one ormore other IGF-1R antagonists. In further embodiments, antigen bindingprotein are administered alone or in combination with other agentsuseful for treating the condition with which the patient is afflicted.Examples of such agents include both proteinaceous and non-proteinaceousdrugs. When multiple therapeutics are co-administered, dosages may beadjusted accordingly, as is recognized in the pertinent art.“Co-administration” and combination therapy are not limited tosimultaneous administration, but also include treatment regimens inwhich an antigen binding protein is administered at least once during acourse of treatment that involves administering at least one othertherapeutic agent to the patient.

Examples of other agents that may be co-administered with an antigenbinding protein are other antigen binding proteins or therapeuticpolypeptides that are chosen according to the particular condition to betreated. Alternatively, non-proteinaceous drugs that are useful intreating one of the particular conditions discussed above may beco-administered with an IGF-1R antagonist.

Combination Therapy

In another aspect, the present invention provides a method of treating asubject with an IGF-1R inhibiting antigen binding protein and one ormore other treatments. In one embodiment, such a combination therapyachieves synergy or an additive effect by, for example, attackingmultiple sites or molecular targets in a tumor. Types of combinationtherapies that can be used in connection with the present inventioninclude inhibiting or activating (as appropriate) multiple nodes in asingle disease-related pathway, multiple pathways in a target cell, andmultiple cell types within a target tissue (e.g., within a tumor). Forexample, an IGF-1R inhibitor of the present invention can be combinedwith a treatment that inhibits IGF-1, promotes apoptosis, inhibitsangiogenesis, or inhibits macrophage. In another embodiment, a targetedagent, that, when used by itself, fails to elicit a therapeuticallydesired effect, could be used to, for example, sensitize cancer cells oraugment treatment effect of other agents. In another embodiment, anIGF-1R inhibitor according to the invention is used in combination witha cytotoxic drug or other targeted agent that induces apoptosis. Inanother embodiment, an IGF-1R inhibitor is used in combination with oneor more agents that inhibit different targets that are involved in cellsurvival (e.g., PKB, mTOR), different receptor tyrosine kinases (e.g.,ErbB1, ErbB2, c-Met, c-kit), or different cell types (e.g., KDRinhibitors, c-fms). In another embodiment, an IGF-1R inhibitor of theinvention is added to the existing standard of care for a particularcondition. Examples of therapeutic agents include, but are not limitedto, gemcitabine, taxol, taxotere, and CPT-11.

In another embodiment, a combination therapy method comprisesadministering to the subject two, three, four, five, six, or more of theIGF-1R agonists or antagonists described herein. In another embodiment,the method comprises administering to the subject two or more treatmentsthat together inhibit or activate (directly or indirectly)IGF-1R-mediated signal transduction. Examples of such methods includeusing combinations of two or more IGF-1R inhibiting antigen bindingprogeins, of an IGF-1R inhibiting antigen binding protein and one ormore other IGF-1, IGF-2, and/or IGF-1R agonists or antagonists (e.g.,IGF-1 and/or IGF-2 binding polypeptides, IGF-1R binding polypeptides,IGF-1 and/or IGF-2 derivatives, anti-IGF-1 and/or IGF-2 antibodies,anti-sense nucleic acids against IGF-1, IGF-2, and/or IGF-1R, or othermolecules that bind to IGF-1, IGF-2, and/or IGF-1R polypeptides ornucleic acids), or of an IGF-1R inhibiting antigen binding protein andone or more other treatments (e.g., surgery, ultrasound, radiotherapy,chemotherapy, or treatment with another anti-cancer agent), asdescribed, for example, in U.S. Pat. No. 5,473,054 (issued Dec. 5,1995), U.S. Pat. No. 6,051,593 (issued Apr. 18, 2000), U.S. Pat. No.6,084,085 (issued Jul. 4, 2000), U.S. Pat. No. 6,506,763 (issued Jan.14, 2003), US Pat. App. Pub. Nos. 03/0092631 (published May 15, 2003),03/0165502 (published Sep. 4, 2003), 03/0235582 (published Dec. 25,2003), 04/0886503 (published May 6, 2004), 05/0272637 (published Dec. 8,2005), PCT Pub. Ser. Nos. WO 99/60023 (published Nov. 25, 1999), WO02/053596 (published Jul. 11, 2002), WO 02/072780 (published Sep. 19,2002), WO 03/027246 (published Mar. 3, 2003), WO 03/020698 (publishedMar. 13, 2003), WO 03/059951 (published Jul. 24, 2003), WO 03/100008(published Dec. 4, 2003), WO 03/106621 (published Dec. 24, 2003), WO04/071529 (published Aug. 26, 2004), WO 04/083248 (published Sep. 30,2004), WO 04/087756 (published Oct. 14, 2004), WO 05/112969 (publishedDec. 1, 2005), Kull et al., 1983, J Biol Chem 258:6561-66, Flier et al.,1986, Proc Natl Acad Sci USA 83:664-668, Conover et al., 1987, J CellPhysiol 133:560-66, Rohlik et al., 1987, Biochem Biophys Res Comm149:276-81, Arteaga et al., 1989, J Clinical Investigation 84:1418-23,Arteaga et al., 1989, Cancer Res 49:6237-41, Gansler et al., 1989,American J Pathol 135:961-66, Gustafson et al., 1990, J Biol Chem265:18663-67, Steele-Perkins et al., 1990, Biochem Biophys Res Comm171:1244-51, Cullen et al., 1992, Mol Endocrinol 6:91-100, Soos et al.,1992, J Biol Chem 267:12955-63, Xiong et al., 1992, Proc Natl Acad SciUSA 89:5356-60, Brunner et al., 1993, Euro J Cancer 29A:562-69,Furlanetto et al., 1993, Cancer Res 53:2522-26, Li et al., 1993, BiochemBiophys Res Comm 196:92-98, Kalebic et al., 1994, Cancer Res 54:5531-34,Lahm et al., 1994, Intl J Cancer 58:452-59, Zia et al., 1996, J CellBiochem Supp 24:269-75, Jansson et al., 1997, J Biol Chem 272:8189-97,Scotlandi et al., 1998, Cancer Res 58:4127-31, Logie et al., 1999, Li etal., 2000, Cancer Immunol Immunotherapy 49:243-52, J Mol Endocrinol23:23-32, De Meyts et al., 2002, Nature Reviews 1:769-83, Hailey et al.,2002, Mol Cancer Therapeutics 1:1349-53, Maloney et al., 2003, CancerResearch 63:5073-83, Burtrum et al., 2003, Cancer Research 63:8912-21,and Karavitaki et al., 2004, Hormones 3:27-36, (each incorporated hereinby reference in its entirety) may be employed in methods andcompositions of the present invention. Furthermore, one or moreanti-IGF-1R antibodies or antibody derivatives can be used incombination with one or more molecules or other treatments, wherein theother molecule(s) and/or treatment(s) do not directly bind to or affectIGF-1R, IGF-1, or IGF-2, but which combination is effective for treatingor preventing a condition, such as cancer or an overgrowth disorder(e.g., acromegaly). In one embodiment, one or more of the molecule(s)and/or treatment(s) treats or prevents a condition that is caused by oneor more of the other molecule(s) or treatment(s) in the course oftherapy, e.g., nausea, fatigue, alopecia, cachexia, insomnia, etc. Inevery case where a combination of molecules and/or other treatments isused, the individual molecule(s) and/or treatment(s) can be administeredin any order, over any length of time, which is effective, e.g.,simultaneously, consecutively, or alternately. In one embodiment, themethod of treatment comprises completing a first course of treatmentwith one molecule or other treatment before beginning a second course oftreatment. The length of time between the end of the first course oftreatment and beginning of the second course of treatment can be anylength of time that allows the total course of therapy to be effective,e.g., seconds, minutes, hours, days, weeks, months, or even years.

In another embodiment, the method comprises administering one or more ofthe IGF-1R antagonists described herein and one or more other treatments(e.g., a therapeutic or palliative treatment), for example, anti-cancertreatments (such as surgery, ultrasound, radiotherapy, chemotherapy, ortreatment with another anti-cancer agent). Where a method comprisesadministering more than one treatment to a subject, it is to beunderstood that the order, timing, number, concentration, and volume ofthe administrations is limited only by the medical requirements andlimitations of the treatment, i.e., two treatments can be administeredto the subject, e.g., simultaneously, consecutively, alternately, oraccording to any other regimen. Examples of agents that can beadministered in combination with the IGF-1R antagonists described hereininclude, but are not limited to, neutrophil-boosting agents,irinothecan, SN-38, gemcitabine, herstatin, or an IGF-1R-bindingherstatin derivative (as described, for example, in U.S. Pat App. No.05/0272637), AVASTIN® (Genentech, South San Francisco, Calif.),HERCEPTIN® (Genentech), RITUXAN® (Genentech), ARIMIDEX® (AstraZeneca,Wilmington, Del.), IRESSA® (AstraZeneca), BEXXAR® (Corixa, Seattle,Wash.), ZEVALIN® (Biogen Idec, Cambridge, Mass.), ERBITUX® (ImcloneSystems Inc., New York, N.Y.), GEMZAR® (Eli Lilly and Co., Indianapolis,Ind.), CAMPTOSAR® (Pfizer, New York, N.Y.), GLEEVEC® (Novartis),SU-11248 (Pfizer), BMS-354825 (Bristol-Myers Squibb), panitumumab(Abgenix, Fremont, Calif./Amgen Inc., Thousand Oaks, Calif.), anddenosumab (Amgen Inc., Thousand Oaks, Calif.).

The following examples, both actual and prophetic, are provided for thepurpose of illustrating specific embodiments or features of the instantinvention and do not limit its scope.

Example 1: Preparation of Antibodies

This example demonstrates a method of preparing antibodies recognizingthe IGF-1 receptor.

IGF-1 receptor polypeptides may be employed as immunogens in generatingmonoclonal antibodies by conventional techniques. It is recognized thatpolypeptides in various forms may be employed as immunogens, e.g., fulllength proteins, fragments thereof, fusion proteins thereof such as Fcfusions, cells expressing the recombinant protein on the cell surface,etc.

To summarize an example of such a procedure, an IGF-1R immunogenemulsified in complete Freund's adjuvant is injected subcutaneously intoLewis rats, in amounts ranging from 10-100 μl. Three weeks later, theimmunized animals are boosted with additional immunogen emulsified inincomplete Freund's adjuvant and boosted every three weeks thereafter.Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay, ELISA (enzyme-linkedimmunosorbent assay), or inhibition of binding of ¹²⁵I-IGF-1 or¹²⁵I-IGF-2 to extracts of IGF-1R-expressing cells. Following detectionof an appropriate antibody titer, positive animals are given a finalintravenous injection of antigen in saline. Three to four days later,the animals are sacrificed, splenocytes harvested, and fused to themurine myeloma cell line AG8653. The resulting hybridoma cell lines areplated in multiple microtiter plates in a HAT selective medium(hypoxanthine, aminopterin, and thymidine) to inhibit proliferation ofnon-fused cells, myeloma hybrids, and spleen cell hybrids.

Hybridoma clones thus generated are screened for reactivity with IGF-1R.Initial screening of hybridoma supernatants utilizes an antibody captureand binding of partially purified ¹²⁵I-IGF-1 receptor. Hybridomas thatare positive in this screening method are tested by a modified antibodycapture to detect hybridoma cells lines that are producing blockingantibody. Hybridomas that secrete a monoclonal antibody capable ofinhibiting ¹²⁵I-IGF-1 binding to cells expressing IGF-1R are thusdetected. Such hydridomas then are injected into the peritoneal cavitiesof nude mice to produce ascites containing high concentrations (>1mg/ml) of anti-IGF-1R monoclonal antibody. The resulting monoclonalantibodies may be purified by ammonium sulfate precipitation followed bygel exclusion chromatography, and/or affinity chromatography based onbinding of antibody to Protein G.

Similar methods can be used to generate human antibodies in transgenicmice. See, e.g., Chen et al., 1993, Internat. Immunol. 5: 647-56; Chenet al., 1993, EMBO J. 12: 821-30; Choi et al., 1993, Nature Genetics 4:117-23; Fishwild et al., 1996, Nature Biotech. 14: 845-51; Harding etal., 1995, Annals New York Acad. Sci.; Lonberg et al., 1994, Nature 368:856-59; Lonberg, 1994, Handbook Experl. Pharmacol. 113: 49-101; Lonberget al., 1995, Internal Rev. Immunol. 13: 65-93; Morrison, 1994, Nature368: 812-13; Neuberger, 1996, Nature Biotech. 14: 826; Taylor et al.,1992, Nuc. Acids Res. 20: 6287-95; Taylor et al., 1994, Internat.Immunol. 6: 579-91; Tomizuka et al., 1997, Nature Genetics 16: 133-43;Tomizuka et al., 2000, Proc. Nat. Acad. Sci. USA 97: 722-27; Tuaillon etal., 1993, Proc. Nat. Acad. Sci. USA 90: 3720-24; Tuaillon et al., 1994,J. Immunol. 152: 2912-20; Russel et al., 2000, Infection and ImmunityApril 2000: 1820-26; Gallo et al., 2000, Eur. J. Immunol. 30: 534-40;Davis et al., 1999, Cancer Metastasis Rev. 18:421-25; Green, 1999, J.Immunol. Methods 231:11-23; Jakobovits, 1998, Advanced Drug DeliveryRev. 31:33-42; Green et al., 1998, J. Exp. Med. 188: 483-95; Jakobovits,1998, Exp. Opin. Invest. Drugs 7: 607-14; Tsuda et al., 1997, Genomics42: 413-21; Mendez et al., 1997, Nature Genetics 15: 146-56; Jakobovits,1996, Weir's Handbook of Experimental Immunology, The Integrated ImmuneSystem Vol. IV, 194.1-194.7; Mendez et al., 1995, Genomics 26: 294-307;Jakobovits, 1994, Current Biol. 4: 761-63; Arbones, 1994, Immunity 1:247-60; Green et al., 1994, Nature Genetics 7: 13-21; Jakobovits et al.,1993, Nature 362: 255-58; Jakobovits et al., 1993, Proc. Nat. Acad. Sci.USA 90: 2551-55.

Example 2: Isolation of Human IGF-1R(ECD)-C3-muIgG1

This example provides a method of making a soluble fragment of IGF-1Ruseful for raising antibodies.

Cloning of pDSRα:huIGF-1R(ECD)-C3-muIgG1Fc

Primers 2830-36: SEQ ID NO: 256) 5′AGCAAGCTTCCACCATGAAGTCTGGCTCCGGAGGAGG 3′ and 2830-38: SEQ ID NO: 257) 5′ATTTGTCGACTTCGTCCAGATGGATGAAGTTTTCAT 3′,were used to amplify the human IGF-1R extracellular domain (1-906) cDNAsequence. The primers included a Kozak translation initiation sequence(underlined above) preceding the start codon, restriction sites forsubsequent subcloning, and a caspace-3 site, which is inserted next tothe extracellular domain C-terminus. PCR was performed on a PerkinElmer2400 (PerkinElmer, Torrance, Calif.) under the following conditions: 1cycle at 95° C. for 2 min, 23 cycles at 95° C. for 30 sec, 58.5° C. for30 sec, and 72° C. for 3 min, and 1 cycle at 72° C. for 10 min. Finalreaction conditions were 1× pfu TURBO® buffer (Stratagene, La Jolla,Calif.), 200 μM dNTPs, 2 μM each primer, 5 U pfu TURBO® (Stratagene) and1 ng template DNA. The PCR product was purified using a ClontechNucleospin Column (Clontech, Palo Alto, Calif.) according to themanufacturers instructions, digested with Hind III and Sal I (Roche,Indianapolis, Ind.) and gel purified. The human IGF-1R insert wasligated into Hind III/Sal I digested pDSRα-muIgG1. Integrity of theinsert was confirmed by DNA sequencing. The sequence of the proteinencoded by the resulting open reading frame (IGF-1R-C3-muFc) is shown inFIG. 10. The final expression vector, pDSRα:huIGF1R(ECD)-C3-muIgG1 Fc,is described in Table 1.

TABLE 1 pDSRα:huIGF1R(ECD)-C3-muIgG1Fc Plasmid Base Pair Number: 11-3496HuIGF1R (Caspase 3 site)-muIgG1Fcatgaagtctggctccggaggagggtccccgacctcgctgtgggggctcctgtttctctccgccgcgctctcgctctggccgacgagtggagaaatctgcgggccaggcatcgacatccgcaacgactatcagcagctgaagcgcctggagaactgcacggtgatcgagggctacctccacatcctgctcatctccaaggccgaggactaccgcagctaccgcttccccaagctcacggtcattaccgagtacttgctgctgttccgagtggctggcctcgagagcctcggagacctcttccccaacctcacggtcatccgcggctggaaactcttctacaactacgccctggtcatcttcgagatgaccaatctcaaggatattgggattacaacctgaggaacattactcggggggccatcaggattgagaaaaatgctgacctctgttacctctccactgtggactggtccctgatcctggatgcggtgtccaataactacattgtggggaataagcccccaaaggaatgtggggacctgtgtccagggaccatggaggagaagccgatgtgtgagaagaccaccatcaacaatgagtacaactaccgctgctggaccacaaaccgctgccagaaaatgtgcccaagcacgtgtgggaagcgggcgtgcaccgagaacaatgagtgctgccaccccgagtgcctgggcagctgcagcgcgcctgacaacgacacggcctgtgtagcttgccgccactactactatgccggtgtctgtgtgcctgcctgcccgcccaacacctacaggtttgagggctggcgctgtgtggaccgtgacttctgcgccaacatcctcagcgccgagagcagcgactccgaggggtttgtgatccacgacggcgagtgcatgcaggagtgcccctcgggcttcatccgcaacggcagccagagcatgtactgcatcccttgtgaaggtccttgcccgaaggtctgtgaggaagaaaagaaaacaaagaccattgattctgttacttctgctcagatgctccaaggatgcaccatcttcaagggcaatttgctcattaacatccgacgggggaataacattgcttcagagctggagaacttcatggggctcatcgaggtggtgacgggctacgtgaagatccgccattctcatgccttggtctccttgtccttcctaaaaaaccttcgcctcatcctaggagaggagcagctagaagggaattactccttctacgtcctcgacaaccagaacttgcagcaactgtgggactgggaccaccgcaacctgaccatcaaagcagggaaaatgtactttgctttcaatcccaaattatgtgtttccgaaatttaccgcatggaggaagtgacggggactaaagggcgccaaagcaaaggggacataaacaccaggaacaacggggagagagcctcctgtgaaagtgacgtcctgcatttcacctccaccaccacgtcgaagaatcgcatcatcataacctggcaccggtaccggccccctgactacagggatctcatcagcttcaccgtttactacaaggaagcaccctttaagaatgtcacagagtatgatgggcaggatgcctgcggctccaacagctggaacatggtggacgtggacctcccgcccaacaaggacgtggagcccggcatcttactacatgggctgaagccctggactcagtacgccgtttacgtcaaggctgtgaccctcaccatggtggagaacgaccatatccgtggggccaagagtgagatcttgtacattcgcaccaatgcttcagttccttccattcccttggacgttctttcagcatcgaactcctcttctcagttaatcgtgaagtggaaccctccctctctgcccaacggcaacctgagttactacattgtgcgctggcagcggcagcctcaggacggctacctttaccggcacaattactgctccaaagacaaaatccccatcaggaagtatgccgacggcaccatcgacattgaggaggtcacagagaaccccaagactgaggtgtgtggtggggagaaagggccttgctgcgcctgccccaaaactgaagccgagaagcaggccgagaaggaggaggctgaataccgcaaagtctttgagaatttcctgcacaactccatcttcgtgcccagacctgaaaggaagcggagagatgtcatgcaagtggccaacaccaccatgtccagccgaagcaggaacaccacggccgcagacacctacaacatcactgacccggaagagctggagacagagtaccctttctttgagagcagagtggataacaaggagagaactgtcatttctaaccttcggcctttcacattgtaccgcatcgatatccacagctgcaaccacgaggctgagaagctgggctgcagcgcctccaacttcgtctttgcaaggactatgcccgcagaaggagcagatgacattcctgggccagtgacctgggagccaaggcctgaaaactccatctttttaaagtggccggaacctgagaatcccaatggattgattctaatgtatgaaataaaatacggatcacaagttgaggatcagcgagaatgtgtgtccagacaggaatacaggaagtatggaggggccaagctaaaccggctaaacccggggaactacacagcccggattcaggccacatctctctctgggaatgggtcgtggacagatcctgtgttcttctatgtccaggccaaaacaggatatgaaaacttcatccatctggacgaagtcgacggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtaaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaa (SEQ ID NO: 258) 3507 to 4391A transcription termination/polyadenylation signal from the α-subunit ofthe bovine pituitary glycoprotein hormone (α-FSH) (Goodwin et al.,1983, Nucleic Acids Res. 11: 6873-82; Genbank Accession Number X00004)4600 to 5163A mouse dihydrofolate reductase (DHFR) minigene containing theendogenous mouse DHFR promoter, the cDNA coding sequences, andthe DHFR transcription termination/polyadenylation signals (Gasser etal., 1982, Proc. Natl. Acad. Sci. U.S.A. 79 6522-6; Nunberg et al.,1980, Cell 19: 355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7;McGrogan et al., 1985, J. Biol. Chem. 260: 2307-14) 6389 to 7246pBR322 sequences containing the ampicillin resistance marker gene andthe origin for replication of the plasmid in E. coli (Genbank AccessionNumber J01749) 7459 to 7802An SV40 early promoter, enhancer and origin of replication (Takebe etal,, 1988, Mol. Cell Biol. 8: 466-72, Genbank Accession Number J02400)7809 to 8065 A translational enhancer element from the HTLV-1 LTR domain(Seiki et al,, 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 3618-22, GenbankAccession Number J02029) 8109 to 8205An intron from the SV40 16S, 19S splice donor/acceptor signals(Okayama and Berg, 1983. Mol. Cell Biol. 3: 280-9, Genbank AccessionNumber J02400)Expression of hu IGF-1R(ECD)-C3-muIgG1 Fc

Fifteen micrograms of linearized expression vectorpDSRα:huIGF1R(ECD)-C3-muIgG1 Fc was transfected into AM-1/D CHOd-cellsusing LT1 lipofection reagent (PanVera Corp., Madison, Wis.), and cellscultured under conditions to allow expression and secretion of proteininto the cell media. Twenty-four colonies were selected after 10-14 dayson DHFR selection medium (Dulbecco's Modified Eagles Medium (Invitrogen)supplemented with 10% dialyzed fetal bovine serum, 1×penicillin-streptomycin (Invitrogen)) and expression levels evaluated bywestern blot. To perform this assay, 0.5 ml of serum free medium wasadded to a single well confluent cells cultured in a 24 well plate(Falcon). The conditioned medium was recovered after 48 hr. Samples forwestern blotting were run in 10% Tris-glycine gel (Novex), and blottedon 0.45 μm Nitrocellulose membrane (Invitrogen), using the MiniTrans-Blot cell (Biorad). The blotted membranes were incubated withrabbit anti-mouse IgG Fc antibody, conjugated with HorseradishPeroxidase (Pierce). The clone expressing the highest level ofIGF-1R(ECD)-C3-muIgG1 Fc was expanded in DHFR selection medium and 2×10⁷cells were inoculated into 50 roller bottles each (Corning) in 250 ml ofhigh-glucose DMEM (Invitrogen), 10% dialyzed FBS (Invitrogen), 1×glutamine (Invitrogen), 1× Non essential amino acids (Invitrogen), 1×sodium pyruvate (Invitrogen). Medium was gassed with 10% CO₂/balance airfor 5 seconds before capping the roller bottle. Roller bottles were keptat 37° C. on roller racks spinning at 0.75 rpm.

When cells reached approximately 85-90% confluency (after approximately5-6 days in culture), growth medium was discarded, cells washed with 100ml PBS and 200 ml production medium was added (50% DMEM (Invitrogen)/50%F12 (Invitrogen), 1× glutamine (Invitrogen), 1× non-essential aminoacids (Invitrogen), 1× sodium pyruvate (Invitrogen), 1.5% DMSO (Sigma)).The conditioned medium was harvested and replaced at one week intervals.The resulting 30 liters of conditioned medium were filtered through a0.45 μm cellulose acetate filter (Corning, Acton, Mass.).

Purification of hu IGF-1R(ECD)-C3-muIgG1 Fc

The resulting filtrate from the conditioned medium was concentrated20-fold using a spiral-wound cartridge (molecular weight cut-off=10kDa), then diluted 1:1 with 3 M KCl, 1 M glycine, pH 9.0 to bring thefinal salt concentration to 1.5 M KCl, 0.5 M glycine, pH 9.0. Thissample was applied to a rProtein A-Sepharose column (Amersham PharmaciaBiotech, Uppsala, Sweden) which had been equilibrated in 1.5 M KCl, 0.5M glycine, pH 9.0. The column was washed with 40 column volumes of thesame buffer, then eluted with 20 column volumes of 0.1 M glycine-HCl, pH2.8. Five-mL fractions were collected and immediately neutralized with 1mL of 1 M Tris-HCl, pH 7.5. Fractions containing huIGF1R(ECD)-C3-muIgGFcwere identified by SDS-PAGE, pooled, and dialyzed againstphosphate-buffered saline. The yield was 2.4 mg/L of conditioned medium.The major protein species detected were the mature α and β chains andmurine Fc, each of which appeared to be properly glycosylated based ontheir elevated and heterogeneous molecular weights. UnprocessedIGF-1R(ECD), as well as glycosylated but not proteolytically cleavedIGF-1R(CED), was also present in the preparation. The shift in bands tohigher molecular weights under non-reducing conditions indicates thatdisulfide linkages joined the α and β chains. Amino-terminal sequencingof the final product indicated that 60% of the protein was correctlyprocessed between the α- and β-chains of IGF-1R(ECD), while 40% remainedunprocessed.

Example 3: Isolation of Human INSR(ECD)-muIgG1

This example presents a method of cloning and expressing a solublefragment of the human insulin receptor.

Cloning of pDSRα:huINSR(ECD)-muIgG1 Fc

Primers 2830-40: SEQ ID NO: 259 5′ AGCAAGCTTCCACCATGGGCACCGGGGGCCGG 3′(Hind III site underlined) and 2830-41: SEQ ID NO: 260 5′ATTTGTCGACTTTTGCAATATTTGACGGGACGTCTAA 3′ (Sal I site underlined)were used to amplify the human INSR extracellular domain (1-929) fromand INSR parental plamid encoding the B form of the INSR splice variant(Ullrich et al., 1985, Nature 313:756-61; Ebina et al., 1985, Cell40:747-58). The primers included a Kozak translation initiation sequencepreceding the start codon and restriction sites for subsequentsub-cloning. PCR was performed on a PerkinElmer 2400 under the followingconditions: 1 cycle at 95° C. for 2 min, 32 cycles at 95° C. for 30 sec,58.5° C. for 30 sec, and 72° C. for 3 min, and 1 cycle at 72° C. for 10min. Final reaction conditions were 1× pfu TURBO® buffer, 200 μM dNTPs,2 μM each primer, 5 Upfu TURBO® (Stratagene) and 10 ng template DNA. ThePCR product was purified using a NUCLEOSPIN® Column (BD BiosciencesClontech, Palo Alto, Calif.) according to the manufacturer'sinstructions, digested with Hind III and Sal I (Roche), and gel purifiedprior to ligation into Hind III/Sal I digested pDSRα-muIgG1. Theintegrity of the insert was confirmed by DNA sequencing. The proteinsequence of the INSR-muFc is shown in FIG. 11. The final expressionvector is described in Table 2.

TABLE 2 Plasmid Base Pair Number: 11-3550 HuINSR-muIgG1Fcatgggcaccgggggccggcggggggcggcggccgcgccgctgctggtggcggtggccgcgctgctactgggcgccgcgggccacctgtaccccggagaggtgtgtcccggcatggatatccggaacaacctcactaggttgcatgagctggagaattgctctgtcatcgaaggacacttgcagatactcttgatgttcaaaacgaggcccgaagatttccgagacctcagtttccccaaactcatcatgatcactgattacttgctgctcttccgggtctatgggctcgagagcctgaaggacctgttccccaacctcacggtcatccggggatcacgactgttctttaactacgcgctggtcatcttcgagatggttcacctcaaggaactcggcctctacaacctgatgaacatcacccggggttctgtccgcatcgagaagaacaatgagctctgttacttggccactatcgactggtcccgtatcctggattccgtggaggataatcacatcgtgttgaacaaagatgacaacgaggagtgtggagacatctgtccgggtaccgcgaagggcaagaccaactgccccgccaccgtcatcaacgggcagtttgtcgaacgatgttggactcatagtcactgccagaaagtttgcccgaccatctgtaagtcacacggctgcaccgccgaaggcctctgttgccacagcgagtgcctgggcaactgttctcagcccgacgaccccaccaagtgcgtggcctgccgcaacttctacctggacggcaggtgtgtggagacctgcccgcccccgtactaccacttccaggactggcgctgtgtgaacttcagcttctgccaggacctgcaccacaaatgcaagaactcgcggaggcagggctgccaccagtacgtcattcacaacaacaagtgcatccctgagtgtccctccgggtacacgatgaattccagcaacttgctgtgcaccccatgcctgggtccctgtcccaaggtgtgccacctcctagaaggcgagaagaccatcgactcggtgacgtctgcccaggagctccgaggatgcaccgtcatcaacgggagtctgatcatcaacattcgaggaggcaacaatctggcagctgagctagaagccaacctcggcctcattgaagaaatttcagggtatctaaaaatccgccgatcctacgctctggtgtcactttccttcttccggaagttacgtctgattcgaggagagaccttggaaattgggaactactccttctatgccttggacaaccagaacctaaggcagctctgggactggagcaaacacaacctcaccaccactcaggggaaactcttcttccactataaccccaaactctgcttgtcagaaatccacaagatggaagaagtttcaggaaccaaggggcgccaggagagaaacgacattgccctgaagaccaatggggacaaggcatcctgtgaaaatgagttacttaaattttcttacattcggacatcttttgacaagatcttgctgagatgggagccgtactggccccccgacttccgagacctcttggggttcatgctgttctacaaagaggccccttatcagaatgtgacggagttcgatgggcaggatgcgtgtggttccaacagttggacggtggtagacattgacccacccctgaggtccaacgaccccaaatcacagaaccacccagggtggctgatgcggggtctcaagccctggacccagtatgccatctttgtgaagaccctggtcaccttttcggatgaacgccggacctatggggccaagagtgacatcatttatgtccagacagatgccaccaacccctctgtgcccctggatccaatctcagtgtctaactcatcatcccagattattctgaagtggaaaccaccctccgaccccaatggcaacatcacccactacctggttttctgggagaggcaggcggaagacagtgagctgttcgagctggattattgcctcaaagggctgaagctgccctcgaggacctggtctccaccattcgagtctgaagattctcagaagcacaaccagagtgagtatgaggattcggccggcgaatgctgctcctgtccaaagacagactctcagatcctgaaggagctggaggagtcctcgtttaggaagacgtttgaggattacctgcacaacgtggttttcgtccccagaaaaacctcttcaggcactggtgccgaggaccctaggccatctcggaaacgcaggtcccttggcgatgttgggaatgtgacggtggccgtgcccacggtggcagctttccccaacacttcctcgaccagcgtgcccacgagtccggaggagcacaggccttttgagaaggtggtgaacaaggagtcgctggtcatctccggcttgcgacacttcacgggctatcgcatcgagctgcaggcttgcaaccaggacacccctgaggaacggtgcagtgtggcagcctacgtcagtgcgaggaccatgcctgaagccaaggctgatgacattgttggccctgtgacgcatgaaatctttgagaacaacgtcgtccacttgatgtggcaggagccgaaggagcccaatggtctgatcgtgctgtatgaagtgagttatcggcgatatggtgatgaggagctgcatctctgcgtctcccgcaagcacttcgctctggaacggggctgcaggctgcgtgggctgtcaccggggaactacagcgtgcgaatccgggccacctcccttgcgggcaacggctcttggacggaacccacctatttctacgtgacagactatttagacgtcccgtcaaatattgcaaaagtcgacggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtaaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaa (SEQ ID NO: 261)3557 to 4441A transcription termination/polyadenylation signal from the α-subunit ofthe bovine pituitary glycoprotein hormone (α-FSH) (Goodwin et al.,1983, Nucleic Acids Res. 11: 6873-82; Genbank Accession Number X00004)4446 to 5586A mouse dihydrofolate reductase (DHFR) minigene containing theendogenous mouse DHFR promoter, the cDNA coding sequences, andthe DHFR transcription termination/polyadenylation signals (Gasser etal., 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al.,1980, Cell 19: 355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7;McGrogan et al., 1985, J. Biol. Chem. 260: 2307-14) 5594 to 6241pBR322 sequences containing the ampicillin resistance marker gene andthe origin for replication of the plasmid in E. coli (Genbank AccessionNumber J01749) 7513 to 7856An SV40 early promoter, enhancer and origin of replication (Takebe etal., 1988, Mol. Cell Biol. 8: 466-72, Genbank Accession Number J02400)7863 to 8119 A translational enhancer element from the HTLV-1 LTR domain(Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 3618-22, GenbankAccession Number J02029) 8163 to 8259An intron from the SV40 16S, 19S splice donor/acceptor signals(Okayama and Berg, 1983. Mol. Cell Biol. 3: 280-9, Genbank AccessionNumber J02400)Expression of hu INSR(ECD)-C3-muIgG1 Fc

AM-1/D CHOd-cells were transfected with 15 μm of linearized expressionvector pDSRα:huINSR(ECD)—muIgG1 Fc using FUGENE™ 6 lipofection reagent(Roche Diagnostics Corp., Indianapolis, Ind.), then cultured underconditions to allow expression and secretion of protein into the cellmedium. Colonies were selected and analyzed as described above.

Purification of hu INSR(ECD)-C3-muIgG1Fc

The filtered conditioned medium containing huINSR(ECD)-muIgGFc wasconcentrated 17-fold using a spiral-wound cartridge (molecular weightcut-off=10 kDa), then diluted 1:1 with 3 M KCl, 1 M glycine, pH 9.0 tobring the final salt concentration to 1.5 M KCl, 0.5 M glycine, pH 9.0.This sample was applied to a rProtein A-Sepharose column (Pharmacia)which had been equilibrated in 1.5 M KCl, 0.5 M glycine, pH 9.0. Thecolumn was washed with 40 column volumes of the same buffer, then elutedwith 20 column volumes of 0.1 M glycine-HCl, pH 2.8. Five-mL fractionswere collected and immediately neutralized with 1-mL of 1 M Tris-HCl, pH7.5. Fractions containing huINSR(ECD)-muIgGFc were identified bySDS-PAGE, pooled, and dialyzed against phosphate-buffered saline. Theyield was 0.9 mg/L of conditioned medium. The major protein species werethe mature α and β chains and murine Fc. Each of these species appearedto be properly glycosylated based on its elevated and heterogeneousmolecular weight. Unprocessed INSR (ECD) as well as glycosylated but notproteolytically cleaved INSR (CED) also was present in the preparation.The shift in bands to higher molecular weights under non-reducingconditions indicated that disulfide linkages joined the α and β chains.Amino-terminal sequencing of the final product indicated that 87% of theprotein was correctly processed between the α- and β-chains ofINSR(ECD), while 13% remained unprocessed.

Example 3: Initial Screen for Anti-IGF-1R phage Fab

This example provides a method of identifying anti-IGF-1R antibodies.

A Target Quest Q Fab library (“the TQ library”; Target Quest,Maastricht, the Netherlands), which was constructed using peripheralblood lymphocytes from four healthy donors and splenic lymphocytes fromone patient with gastric carcinoma, was obtained. The library diversitywas 3.7×10¹⁰ clones, containing 3×10⁹ heavy chains. The source,screening methods, and characterization of the library have beenpublished (de Haard et al, 1999, J Biol Chem 274:18218-30). Dynabeads(200 μl) M-450 Uncoated (catalog #140.02, Dynal, Lake Success, N.Y.)were washed 3 times with PBS, resuspended in 200 μl of IGF1R(ECD)-C3-mFcto a concentration of 0.5 μM in PBS, and incubated at 4° C. on a rotatorovernight. The IGF-1R(ECD)-C3-mFc coated beads were washed 3× with 1 mlof 2% non-fat dry milk (M) in PBS (2% MPBS), and then blocked with 1 mlof 2% MPBS at room temperature for 1 hour. In parallel, 750 μl of the TQlibrary (4×10¹² pfu) was preblocked by mixing with 250 μl 8% MPBS atroom temperature for 30 minutes to 1 hour. 500 μl of blocked beads weretransferred into another microfuge tube and separated from the blockingsolution on a magnetic separator. The preblocked phage mixture was addedto the blocked beads and incubated for 90 minutes on a rotator at roomtemperature. Bead-bound phage were separated from the unbound phage, andthen washed 6× with 1 ml 2% MPBS/0.1% Tween 20, 6× with 1 ml PBS/0.1%Tween 20, 2× with PBS with a change of tubes between different washsolutions. Bound phage was eluted with 1 ml of 0.1 M TEA (pH11) for 10minutes, then immediately separated from the beads and neutralized with0.5 ml of 1 M Tris.HCl. The eluted phage pool was mixed with 4 ml 2× YTbroth (10 g yeast extract, 16 g bacto-tryptone, 5 g NaCl per liter ofwater) and 5 ml of TG1 bacterial culture (O.D.₅₉₀ about 0.5) in a 50-mlconical tube. The infection mixture was incubate at 37° C. in anincubator for 30 min., then centrifuged at 3500 rpm for 20 min. The cellpellet was resuspended in 1500 μl 2× YT-CG broth and 300 μl were spreadon each of five 2× YT-CG (2× YT broth containing 100 μg/ml carbenicillinand 2% glucose) plates. After 20 hours of incubation at 30° C., 4 ml of2× YT-AG were added to each plate and the cells were recovered with cellscraper from the plates. This step was repeated three times. A smallportion of the recovered cells was used for phage rescue (see below).The remaining cell suspension was centrifuged at 3500 rpm for 20 min.The cell pellet was suspended into an amount of 50% glycerol roughlyhalf the volume of the pellet size and stored at −80° C.

In order to rescue phage, the plated-amplified cell suspension was usedto inoculate 40 ml of 2× YT-CG to an OD₅₉₀ of about 0.05. The culturewas incubated at 37° C. on a shaker to OD₅₉₀ 0.5. The log phase culturewas infected with M13KO7 helper phage (GIBCO BRL, Gaithersburg, Md.,catalog #18311-019, 1.1×10¹¹ pfu/ml) at M.O.I. 20 followed by incubationat 37° C. for 30 min. The infected cells were centrifuged at 4000 rpmfor 20 min. The cell pellet was re-suspended in 200 ml of 2× YT-CK (100μg/ml carbenicillin and 40 μg/ml kanamycin) and transferred to two250-ml flasks and incubated at 30° C. with shaking at 270 rpm for 20hours. The over-night culture was centrifuged at 4000 rpm for 20 min toremoval cell debris. The centrifugation was repeated to ensure theremoval of cell debris. About ⅕ volume of PEG solution (20% PEG 8000,2.5 M NaCl) was added to the supernatant to precipitate the phageparticles. The mixture was incubated on ice for at least 1 hour,followed by centrifugation at 4000 rpm for 20 min to collect theprecipitated phage particles. The phage pellet was re-suspended into 1ml of PBS and transferred to a microfuge tube. The phage suspension wasleft on ice for 1 hour to allow complete suspension of phage particles,and clarified by centrifugation at 14,000 rpm for 2 min to remove theresidual cell debris. Phage precipitation step was repeated. The finalphage pellet was suspended into PBS after clarification. The rescuedphage suspension was used in the next round of selection.

Four rounds of selection were performed that included alterations ofvarious standard binding parameters. The second round of selection wasidentical to the first round of selection. Variations in input phagenumber and elution reagent were introduced in rounds three and four. Forthe round three selection, 5×10¹¹ pfu of phages were selected and boundphages were eluted either with 1 μM IGF-1 (catalog #13769, Sigma, St.Louis, Mo.) or with a 1 μM concentration of a chimeric αIR3-huFcantibody to yield two round-three pools, TQ4-3IS and TQ4-3CA. Round fourselection was carried out on rescued phage pools from both round threepools. Two rounds of negative selection with mouse IgG Fc-coatedDYNABEADS® (Dynal Biotech, Oslo, Norway) were included to remove mouseFc binders prior to actual IGF-1R selection. The incubation time fornegative selection was 30 minutes each. 3.78×10¹¹ pfu of TQ4-3IS pooland 3.75×10¹² pfu of TQ4-3CA pool were selected separately. Bound phagewere eluted with 1 μM IGF-2 (catalog #12526, Sigma, St. Louis, Mo.) toyield two round-4 pools, TQ4-4IS12 and TQ4-4CAI2. The sequence of about96-192 phage DNA inserts was determined at each elution step.

In some cases, a secondary screen was done. Phagemid DNA mixtures of thetotal TQ library, and the selected phage amplified after several roundsof selection against IGF-1R, were prepared using a DNA Maxiprep kitaccording to the manufacturer's instructions (Qiagen, Valencia, Calif.).All four DNA preparations were digested with Asc I and EcoR I (NewEngland Biolab, Beverly, Mass.). The resulting two Asc I/EcoR Ifragments were separated on preparative 0.5% agarose gels. The 2.1 kbfragments containing heavy chains were gel purified from the IGF-1Rselected phage. The 3.9 kb fragments containing the light chains andpCES 1 vector portion were gel purified from the total TQ library DNA.The 2.1 kb fragments were ligated to the 3.9 kb fragments from the DNAsample of TQ library in 3:1 ratio. The ligated DNA was precipitated andused to transform TG1 cells by electroporation. The library size of theresulted light chain shuffled secondary library was 8.8×10⁸. Aftersequencing 96 randomly picked clones, 76 unique light chain sequenceswere obtained, indicating that the attempt to shuffle light chains wassuccessful.

The binding, washing and elution condition for screening the light chainshuffle library were essentially the same as decribed for the intialscreen. However, several variations were included to increase selectionpressure for amplification of IGF-1R binders with higher affinities,especially those with significantly slower off-rates. These parameterswere: higher number of input phage (2-2.7×10¹³ pfu), smaller bead volume(100 μl for round one, 50 μl for round two, and 25 μl for round three),and extended specific elution time up to 20 hours. Elution buffers were0.1 M TEA for round one (RD1), 1 μM IGF-1 in 0.4% MPBS for RD2 and 1 μMIGF-1 or IGF-2 in 0.4% MPBS for RD3. In RD2 and RD3, binders that wereeluted in 15 min or 2 hours were discarded. Elution was continued andeluted phages were collected after 8-10 hours and again after 20 hours.

Phage Fab ELISA Screen

In 96-well 2-ml deep-well blocks, 480 μl/well 2× YT-CG broth wasinoculated with 20 μl of overnight cultures of the individual clones,then incubated at 37° C., 300 rpm for 3 hours. To each well, 50 μl of1:3 diluted M13KO7 helper phage were added to infect the cells. Theblock was incubated at 37° C. without shaking for 30 minutes, and thenshaken gently for another 30 minutes at 150 rpm. The block wascentrifuged at 3600 rpm for 20 minutes to pellet the infected cells. Thecell pellet in each well was suspended into 480 μl of 2× YT-CK (2× YTbroth containing 100 μg/ml carbenicillin and 40 μg/mlkanamycin), andincubated at 30° C. overnight for about 20 hours. The cell debris wasseparated by centrifugation at 3600 rpm for 20 minutes. The rescuedphage supernatant was used in the phage ELISA to check forIGF-1R-specific, INSR-cross reactive, or mouse Fc binding of individualclones.

Three sets of Nunc MaxiSorb Immunoplates were coated with 100 μl/well ofIGF-1R-C3-mFc at 5 μg/ml, INSR-mFc at 5 μg/ml, or mouse IgG1 (catalog#010-0103, Rockland, Gilbertsville, Pa.) at 2 μg/ml in PBS,respectively, at 4° C. overnight. The coated plates were washed 3× with300 μl/well of PBS. The washed plates were blocked with 300 μl/well 2%MPBS at room temperature for one hour. Meanwhile, rescued phages ofindividual clones were pre-blocked by mixing 170 μl of rescued phagewith 170 μl of 4% MPBS. The blocked plates were washed 5× with 300μl/well TBST (TBS: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl;Tween-20. 0.1%). 100 μl/well of pre-blocked phage dilutions weredistributed to each set of coated plate, which were incubated at roomtemperature on a rocker for 90 minutes. The plates were washed 5× with300 μl/well TBST. 100 μl/well of anti-M13-HRP in 2% MPBS (1:3000dilution, catalog number 27-9421-01, Amersham Pharmacia Biotech) weredistributed, and plates were incubated at room temperature on rocker forone hour. The plates were washed 5× with 300 μl/well TBST. 100 μl/wellof the substrate 1-Step™ ABTS (Pierce Biotechnology, Rockford, Ill.,catalog number 37615) were added. Plates were incubated for one hour.OD₄₀₅ was measured for signal detection.

The phage displayed antibodies exhibited essentially no crossreactivitywith the insulin receptor and murine Fc domain. The signal observed inthe IGF-1R ELISA is therefore specific for the IGF-1R extracellulardomain. Results from similar assays for four of the phage-displayedantibodies are shown in FIG. 14.

The DNA inserts of IGF-1R positive, INSR and mu IgG1 negative, cloneswere sequenced. Fifty-two unique Fab sequences were identified, havingthe following combinations of light chain and heavy chain variabledomain sequences: L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9,L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18,L19H19, L20, H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26,L27H27, L28H28, L29H29, L30H30, L31H31, L32H32, L33H33, L34H34, L35H35,L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44,L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, and L52H52,wherein “Lx” indicates light chain variable domain number “x” and “Hx”indicates heavy chain variable domain number “x.” FIG. 1 presents thepolynucleotide sequences of each of these light and heavy variabledomains. FIGS. 2 and 3 present the corresponding amino acid sequences.

Example 4: Subcloning of V_(H) and V_(L) into IgG1 Expression Vectors

This example presents a method of subcloning the previously identifiedvariable domain sequences into an IgG1 expression vector.

Construction of pDSRα20 and pDSRα20:hIgG1C_(H)

The pDSRα20:hIgG1C_(H) expression vector (WO 90/14363) was a derivativeof pDSR19:hIgG1C_(H) (see U.S. Provisional Patent Application No.60/370,407, filed Apr. 5, 2002, “Human Anti-OPGL Neutralizing AntibodiesAs Selective OPGL Pathway Inhibitors,” incorporated herein by referencein its entirety). The pDSRα19:hIgG1C_(H) plasmid encoded a rat variableregion/human constant region IgG1 (rVh/hCh1). The plasmid wasconstructed by the three-piece ligation of Xba I and BsmB I terminatedrat antibody variable region PCR product, the human IgG1 constant region(C_(H1), hinge, C_(H2) and C_(H3) domains) derived by Sal I cleavage andgel isolation of the BsmB I and Sal I fragment from the linear plasmidpDSRα19:hIgG1 C_(H) (Hind III and BsmB I ends) and a linearized pDSRα19with Xba I and Sal I ends. pDSRα20 was produced by changing nucleotide2563 in pDSRα19 from a guanosine to an adenosine by site directedmutagenesis. The heavy chain expression vector, pDSRα20:hIgG1C_(H) ratvariable region/human constant region IgG1 (rVh/hChl), is 6163 basepairs and contains the 7 functional regions described in Table 3.

TABLE 3 Plasmid Base Pair Number: 2 to 881A transcription termination/polyadenylation signal from the α-subunit ofthe bovine pituitary glycoprotein hormone (α-FSH) (Goodwin et al.,1983, Nucleic Acids Res. 11: 6873-82; Genbank Accession Number X00004)882 to 2027A mouse dihydrofolate reductase (DHFR) minigene containing theendogenous mouse DHFR promoter, the cDNA coding sequences, andthe DHFR transcription termination/polyadenylation signals (Gasser etal., 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al.,1980, Cell 19: 355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7;McGrogan et al., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947pBR322 sequences containing the ampicillin resistance marker gene andthe origin for replication of the plasmid in E. coli (Genbank AccessionNumber J01749) 3949 to 4292An SV40 early promoter, enhancer and origin of replication (Takebe etal., 1988, Mol. Cell Biol. 8: 466-72, Genbank Accession Number J02400)4299 to 4565 A translational enhancer element from the HTLV-1 LTR domain(Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 3618-22, GenbankAccession Number J02029) 4574 to 4730An intron from the SV40 16S, 19S splice donor/acceptor signals(Okayama and Berg, 1983. Mol. Cell Biol. 3: 280-9, Genbank AccessionNumber J02400) 4755 to 6158The rVh/hCh1 heavy chain cDNA between the Xbal and Sall sites. Thisheavy chain fragment sequence is shown below (SEQ ID NO: 262) withthe sequences of the restriction sites underlined:   XbaI TCTAG ACCACCATGG ACATCAGGCT CAGCTTAGTTTTCCTTGTCC TTTTCATAAA AGGTGTCCAG TGTGAGGTAGAACTGGTGGA GTCTGGGGGC GGCTTAGTAC AACCTGGAAGGTCCATGACA CTCTCCTGTG CAGCCTCGGG ATTCACTTTCAGAACCTATG GCATGGCCTG GGTCCGCCAG GCCCCAACGAAGGGTCTGGA GTGGGTCTCA TCAATTACTG CTAGTGGTGGTACCACCTAC TATCGAGACT CCGTGAAGGG CCGCTTCACTATTTTTAGGG ATAATGCAAA AAGTACCCTA TACCTGCAGATGGACAGTCC GAGGTCTGAG GACACGGCCA CTTATTTCTGTACATCAATT TCGGAATACT GGGGCCACGG AGTCATGGTC  BsmB1ACCGTCTCTA GTGCCTCCAC CAAGGGCCCA TCGGTCTTCCCCCTGGCACC CTCCTCCAAG AGCACCTCTG GGGGCACAGCGGCCCTGGGC TGCCTGGTCA AGGACTACTT CCCCGAACCGGTGACGGTGT CGTGGAACTC AGGCGCCCTG ACCAGCGGCGTGCACACCTT CCCGGCTGTC CTACAGTCCT CAGGACTCTACTCCCTCAGC AGCGTGGTGA CCGTGCCCTC CAGCAGCTTGGGCACCCAGA CCTACATCTG CAACGTGAAT CACAAGCCCAGCAACACCAA GGTGGACAAG AAAGTTGAGC CCAAATCTTGTGACAAAACT CACACATGCC CACCGTGCCC AGCACCTGAACTCCTGGGGG GACCGTCAGT CTTCCTCTTC CCCCCAAAACCCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCACATGCGTGGTG GTGGACGTGA GCCACGAAGA CCCTGAGGTCAAGTTCAACT GGTACGTGGA CGGCGTGGAG GTGCATAATGCCAAGACAAA GCCGCGGGAG GAGCAGTACA ACAGCACGTACCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGGCTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAGCCCTCCCAGC CCCCATCGAG AAAACCATCT CCAAAGCCAAAGGGCAGCCC CGAGAACCAC AGGTGTACAC CCTGCCCCCATCCCGGGATG AGCTGACCAA GAACCAGGTC AGCCTGACCTGCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGAGTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACCACGCCTCCCG TGCTGGACTC CGACGGCTCC TTCTTCCTCTATAGCAAGCT CACCGTGGAC AAGAGCAGGT GGCAGCAGGGGAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCACAACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA   SalI AATGATAAGT CGAC

The linear plasmid pDSRα20:hIgG1C_(H) was prepared by digesting thepDSR20: rat variable region/human constant region IgG1 plasmid with therestriction enzymes Xba I and BsmB I to remove the rat variable regionand purified using a QIAquick Gel Extraction kit. The linear plasmidpDSRα20:hIgG1 C_(H) containing the 1.0 kbp human IgG1 constant regiondomain was used to accept anti-IGF-1R variable heavy chain codingsequences.

Construction of the Anti-IGF-1R IgG1 Heavy Chain Expression Clones

The sequence coding for the anti-IGF-1R variable region of the heavychains was amplified from phagemid DNA with complementaryoligonucleotide primers. Primers for polymerase chain reaction (PCR)were designed to incorporate a Hind III site, Xba I site, Kozak sequence(CCACC) and signal sequence (translated peptide isMDMRVPAQLLGLLLLWLRGARC; SEQ ID NO:263) onto the 5′ end of the variableregion, while a BsmB I site was added onto the 3′ end of the PCRproduct. The PCR products were digested with Xba I and BsmB I, and thencloned into the Xba I-BsmB I linear pDSRα20:hIgG1C_(H) expression vectorcontaining the human IgG1 constant region (FIG. 13). The finalexpression vectors contained the seven functional regions described inTable 4.

TABLE 4 Plasmid Base Pair Number: 2 to 881 A transcriptiontermination/polyadenylation signal from the α-subunit of the bovinepituitary glycoprotein hormone (α-FSH) (Goodwin et al., 1983, NucleicAcids Res. 11: 6873-82; Genbank Accession Number X00004) 882 to 2027 Amouse dihydrofolate reductase (DHFR) minigene containing the endogenousmouse DHFR promoter, the cDNA coding sequences, and the DHFRtranscription termination/polyadenylation signals (Gasser et al., 1982,Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al., 1980, Cell 19:355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7; McGroganet al., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947 pBR322 sequencescontaining the ampicillin resistance marker gene and the origin forreplication of the plasmid in E. coli (Genbank Accession Number J01749)3949 to 4292 An SV40 early promoter, enhancer and origin of replication(Takebe et al., 1988, Mol. Cell Biol. 8: 466-72, Genbank AccessionNumber J02400) 4299 to 4565 A translational enhancer element from theHTLV-1 LTR domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:3618-22, Genbank Accession Number J02029) 4574 to 4730 An intron fromthe SV40 16S, 19S splice donor/acceptor signals (Okayama and Berg, 1983.Mol. Cell Biol. 3: 280-9, Genbank Accession Number J02400) 4755 to 6185The heavy chain IgG1 cDNA between the Xbal and Sall sitesConstruction of the anti-IGF-1R IgG1 Variable Chain Expression Clones.

The light chains encoded in anti-IGF-1R phage were either kappa orlambda class. They were cloned using one of two approaches.Complementary primers were designed to add a Hind III site, an Xba Isite, Kozak sequence (CCACC) and signal sequence (translated peptide isMDMRVPAQLLGLLLLWLRGARC, SEQ ID NO:264) were added to the 5′ end of thecoding region. Those chains that had error-free coding regions werecloned as full-length products. The full-length light chains were clonedas Xba I and Sal I fragments into the expression vector pDSRα20. Thefinal expression vectors contained the seven functional regionsdescribed in Table 5.

TABLE 5 Plasmid Base Pair Number: 2 to 881 A transcriptiontermination/polyadenylation signal from the α-subunit of the bovinepituitary glycoprotein hormone (α-FSH) (Goodwin et al., 1983, NucleicAcids Res. 11: 6873-82; Genbank Accession Number X00004) 882 to 2027 Amouse dihydrofolate reductase (DHFR) minigene containing the endogenousmouse DHFR promoter, the cDNA coding sequences, and the DHFRtranscription termination/polyadenylation signals (Gasser et al, 1982,Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al., 1980, Cell 19:355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7; McGrogan etal., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947 pBR322 sequencescontaining the ampicillin resistance marker gene and the origin forreplication of the plasmid in E. coli (Genbank Accession Number J01749)3949 to 4292 An SV40 early promoter, enhancer and origin of replication(Takebe et al., 1988, Mol. Cell Biol. 8: 466-72, Genbank AccessionNumber J02400) 4299 to 4565 A translational enhancer element from theHTLV-1 LTR domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. U. S. A.80: 3618-22, Genbank Accession Number J02029) 4574 to 4730 An intronfrom the SV40 16S, 19S splice donor/acceptor signals (Okayama and Berg,1983, Mol. Cell Biol. 3: 280-9, Genbank Accession Number J02400) 4755 to5485 The kappa light chain cDNA between the Xbal and Sall sites

Some kappa clones had errors in their constant regions when compared tonatural human constant region sequence. To eliminate thesediscrepancies, the kappa variable region was amplified with a primerthat would introduce an Xba I site into the 5′ end and a BsmB I siteinto the 3′ end. This fragment was then ligated along with a human kappaconstant region (FIG. 13) with a compatible BsmB I on the 5′ end and a3′Sal I ends into pDSRα20 with Xba I and Sal I ends.

Example 5: Transient Expression of Antibodies

This example provides a method of transiently expressing anti-IGF-1Rantibodies.

The antibodies were expressed transiently in serum-free suspensionadapted 293T cells. All transfections were performed as 250 mL cultures.Briefly, 1.25×10⁸ cells (5.0×10⁵ cells/mL×250 mL) were centrifuged at2,500 RPM for 10 minutes at 4° C. to remove the conditioned medium. Thecells were resuspended in serum-free DMEM and centrifuged again at 2,500RPM for 10 minutes at 4° C. After aspirating the wash solution, thecells were resuspended in growth medium [DMEM/F12 (3:1)+1×Insulin-Transferrin-Selenium Supplement+1× Pen Strep Glut+2 mML-Glutamine+20 mM HEPES+0.01% Pluronic F68] in a 500 ml, spinner flaskculture. The spinner flask culture was maintained on magnetic stir plateat 125 RPM which was placed in a humidified incubator maintained at 37°C. and 5% CO₂. The plasmid DNA was incubated with the transfectionreagent in a 50 mL conical tube. The DNA-transfection reagent complexwas prepared in 5% of the final culture volume in serum-free DMEM. Onemicrogram of plasmid DNA per milliliter of culture was first added toserum-free DMEM, followed by 1 μl X-TremeGene RO-1539/mL culture. Thecomplexes were incubated at room temperature for approximately 30minutes and then added to the cells in the spinner flask. Thetransfection/expression was performed for 7 days, after which theconditioned medium was harvested by centrifugation at 4,000 RPM for 60minutes at 4° C.

If the initial transfection failed to yield the required 100 μg purifiedantibody, those clones were re-expressed in roller bottles. Thesetransfections used 293T adherent cells grown and maintained in DMEMsupplemented with 5% FBS+1× Non-Essential Amino Acids+1× Pen StrepGlut+1× Sodium Pyruvate. Approximately, 4-5×10⁷ 293 T cells were seededin a 850 cm² roller bottles overnight. The previously seeded cells werethen transfected the following day using FUGENE™ 6 transfection reagent.The DNA—transfection reagent mixture was prepared in approximately in6.75 mL serum-free DMEM. 675 μl FUGENE™ 6 transfection reagent was firstadded, followed by 112.5 μg plasmid DNA. The complex was incubated atroom temperature for 30 minutes. The entire mixture was then added to aroller bottle. The roller bottle was infused with a 5% CO₂ gas mixture,capped tightly and placed in a 37° C. incubator on a roller rackrotating at 0.35 RPM. The transfection was performed for 24 hours afterwhich the medium was replaced with 100 mL DMEM+1×Insulin-Transferrin-Selenium Supplement+1× Pen Strep Glu+1×Non-Essential Amino Acids+1× Sodium Pyruvate. Typically, 2-3 harvests(100 ml) were obtained from each roller bottle at a 48 hr interval. Theharvested serum-free conditioned medium was pooled together andcentrifuged at 4,000 RPM for 30 minutes at 4° C.

Example 6: Anti-IGF-1R Antibody Small-Scale Purification

This example provides a method of purifying anti-IGF-1R antibodies on asmall scale.

Conditioned medium was filtered through a 0.45 μm cellulose acetatefilter and concentrated approximately 8-fold using a Vivaflow 200 50 Ktangential flow membrane (Vivascience, Goettingen, Germany). rProtein ASEPHAROSE™ Fast Flow resin (Amersham Biosciences, Piscataway, N.J.) waswashed with phosphate buffered saline (2.7 mM potassium chloride, 138 mMsodium chloride, 1.5 mM potassium phosphate, and 8.1 mM sodiumphosphate, pH 7.4) (PBS) four times then directly applied to theconcentrated media. The amount of resin used was based on antibodyconcentration determined by ELISA where 1 μl of resin was used per 5 μgantibody. The medium was incubated overnight at 4° C. with gentleagitation. The resin was centrifuged at 500 g for 10 min. at 4° C. Thesupernatant was decanted as the unbound fraction. The resin was washedwith PBS four times for one minute at room temperature with gentleagitation, each time collecting the resin by centrifugation at 500 g for10 min. at 4° C. The antibody was eluted by incubating the resin with1.5 volumes of 0.1 M glycine pH 3.0 for 10 min. at room temperature. Theresin was centrifuged at 500 g for 10 min. at 4° C. and the supernatantdecanted as eluted antibody. The elution step described above wasrepeated for a total of three elutions; each time the eluted materialwas neutralized with 0.04 volumes of 1.0 M tris-HCl, pH 9.2. The samplewas filtered through a 0.2 μm cellulose acetate filter. Proteinconcentration was determined by the Bradford method using the Bio-RadProtein Assay (Bio-Rad Laboratories, Hercules, Calif.) as per thesupplied instructions using Human IgG (Sigma-Aldrich, St. Louis, Mo.) asa standard. The sample was compared to a Human IgG1, K standard(Sigma-Aldrich, St. Louis, Mo.) using a 4-20% tris-glycine SDSpolyacrylamide gel (SDS-PAGE) gel stained with Coomassie brilliant bluedye. No contaminating protein was visible in these preparations.

Example 7: Isolation of Stable CHO Clones Expressing Antibodies

This example provides a method for isolating stable CHO cell linesexpressing anti-IGF-1R antibodies.

Stable expression of TQ11C, TQ25, TQ 58 and TQ59 IgG1 was achieved byco-transfection of AM1-D CHO cells (U.S. Pat. No. 6,210,924,incorporated herein by reference in its entirety) with pDSRα20 heavy andlight chian IgG1 expression constructs. The plasmid transfections wereperformed using LF2000 (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. Briefly, 4×106AM1-D CHO cells were plated24 hours prior to transfection, in 100 mm diameter FALCON™ plastic petridishes (BD Falcon, Franklin Lakes, N.J.) in 10 ml of Dulbecco's ModifiedEagles Medium (Invitrogen) supplemented with 5% fetal bovine serum, 1×penicillin-streptomycin and glutamine (Invitrogen), non-essential aminoacids (Invitrogen), sodium pyruvate, and HT (0.1 mM sodiumhypoxanthine,16 nM thymidine; Invitrogen). Approximately 15 mg of each pDSRα21-lightchain and heavy chain plasmid DNA were linearized using Pvu I (NewEngland Biolabs) and diluted in 2 ml of OPTI-MEM® (Invitrogen). Thediluted plasmids were mixed with 75 μl of LIPOFECTAMINE™ 2000 (LF2000;GIBCO/BRL) diluted in 2 ml of OPTI-MEM® and the mixture was incubatedfor 20 min at room temperature. The following day fresh growth mediumwas added. The cells were cultured in complete growth medium for 48hours, then plated in HT-selection medium in 1:20 and 1:50 dilutions.Approximately 2 weeks after transfection, 12-24 visible colonies werepicked into 24-well plates, using the sterile cloning discs (RPI). Theclones expressing the highest level of TQ11C, TQ25, TQ58 and TQ59 IgG1were identified by western immunoblot analysis. To perform this assay,0.5 ml of serum free medium was added to a single-well confluent cellscultured in a 24 well plate (BD Falcon). The conditioned medium wasrecovered after 24 hr, and 10 μl of CM was mixed with an equal volume ofloading buffer to run a 10% Tris-Glycine polyacrylamide protein gel(Invitrogen). The gel was transferred to a 0.45 μm pore sizenitrocellulose membrane (Invitrogen), and western blot analysis was doneusing 1:1000 dilution of goat anti-human IgG Fc ImmunoPure antibody(Pierce Biotechnology, Inc., Rockford, Ill.) and ECL as detection agent.

Example 8: Mid-Scale Expression of Antibodies

This example provides a method of expressing anti IGF-1R antibodiesexpressed by stable CHO cell lines.

The CHO cell lines made according to Example 7 were expanded to T-175tissue culture flasks (Falcon) for scale-up expression. A confluent T175flask (approximately 2-3×107 cells) was used to seed 3-850 cm2 rollerbottles (Corning Life Sciences, Acton, Mass.), and three confluentroller bottles (approximately 1-2×108 cells per roller bottle) were usedto seed 30 rollers in 250 ml of high-glucose DMEM (Invitrogen), 10%dialyzed FBS (Invitrogen), 1× glutamine (Invitrogen), 1× non-essentialamino acids (Invitrogen), 1× sodium pyruvate (Invitrogen). Medium wasinfused with 10% CO₂/balance air for 5 seconds before capping the rollerbottle. Roller bottles were incubated at 37° C. on roller racks spinningat 0.75 rpm.

When cells reached approximately 85-90% confluency (approximately 5-6days in culture), the growth medium was discarded, the cells were washedwith 100 ml PBS, and 200 ml production medium was added (50% DMEM(Invitrogen)/50% F12 (Invitrogen), 1× glutamine (Invitrogen), 1×non-essential amino acids (Invitrogen), 1× sodium pyruvate (Invitrogen),1.5% DMSO (Sigma). Conditioned medium was harvested every seven days fora total of four harvests.

Conditioned medium was filtered through a 0.45 μm cellulose acetatefilter and concentrated approximately 10-fold using a Sartorius SartoconSlice Disposable 30 K tangential flow membrane (Sartorius AG,Goettingen, Germany). The concentrated material was applied to a 10 mlrProtein A Sepharose column at 4° C. and the flowthrough was collectedas the unbound fraction. The column was washed with four column volumesof PBS. The bound sample was eluted with approximately four columnvolumes of 0.1 M glycine pH 3.0. The eluate peak was collected andneutralized with 0.04 volumes of 1.0 M tris-HCl, pH 9.2. The eluate wasdialyzed against 150 volumes of PBS overnight at 4° C. The sample wasfiltered through a 0.2 μm cellulose acetate filter and proteinconcentration was measured by determining the absorbance at 280 nm usingan extinction coefficient of 14,000 M-1. The sample was compared to aHuman IgG1, K standard (Sigma-Aldrich, St. Louis, Mo., USA) using a4-20% tris-glycine SDS-PAGE gel stained with Coomassie brilliant bluestain. Endotoxin levels in each antibody prepration was determined usingthe Pyrotell Limulus Amebocyte Lysate Assay (Associates of Cape Cod,Inc., Falmouth, Ma) as per the supplied instructions.

Example 9: ORIGEN® Dose Response Competition Assays

This example provides methods for testing the ability of an antibody toblock ligand binding to IGF-1R.

An ORIGEN® binding assay was used to determine whether TQ11C, TQ25, TQ58 and TQ59 IgG1 antibodies could block ligand binding to IGF-1R usingprocedures provided by the manufacturer (Igen, Inc., Gaithersburg, Md.).To label IGF-1 and IGF-2 with ruthenium, lyophilized proteins weredissolved into PBS to give a 1.0 mg/ml solution. Label (ORI-TAG-NHSester from Igen, Cat #110034) was added to the protein at a molar ratioof 5:1 (label: protein) from a label stock of 5 mg/ml in DMSO. Themixture was incubated at room temperature (20-22° C.) for 1 hr in thedark then treated with 20 μl 2 M glycine for 10 min at room temperature.The labeled protein was separated from the free label by application toan Amersham Biosciences NAP-5 column (Amersham Biosciences, Piscataway,N.J.) equilibrated in PBS and 0.33 ml fractions collected. The proteinconcentration of the fractions was determined by Micro BCA Protein Assay(Pierce Biotechnology, Inc., Rockford, Ill.). Fractions two and threecontained significant protein and were combined. The amount ofincorporated ruthenium label was assessed using the following formula:ruthenium tris-bipyridyl compound (Ru(bpy)₃ ²) labeling of IGF-1 andIGF-2.

Dynal M450 paramagnetic beads coated with sheep anti-mouse IgG was usedas the solid support phase for the IGF-1R(ECD)-C3-muFc. The M450 beadswere prepared for receptor loading by washing three times with assaybuffer containing 1×PBS, 0.05% TWEEN™ 20 (ICI Americas, Inc., WilmingtonDel.) 0.1% BSA, 0.01% sodium azide. The IGF-1R(ECD)-C3-muFc was boundfor 1 hr at a ratio of 50 ng receptor per 1×10⁶ M450 beads in a volumeof 25 μl assay buffer. To generate dose response data, the antibodies orunlabeled IGF-1 and IGF-2 factors were added at increasingconcentrations (10⁻¹¹ M to 10⁻⁶ M) simultaneously with 1 nM Ru-IGF-1 or2 nM Ru-IGF-2. The final reaction volume was 100 μl. After incubation atroom temperature in the dark for 2 hr, an M8 Analyzer (Igen) was used toremove free ruthenium labeled ligand and determine the amount of ligandbound to receptor. The data were expressed as the percent of totalligand bound minus background remaining after competition with excessunlabeled growth IGF1 or IGF-2. Competition curves were generated withGraphPad Prism software (GraphPad Software, San Diego, Calif.) using asingle component equilibirium model. Essentially all (>98%) binding wascompeted with excess unlabeled growth factors. The positive controlantibodies in the binding analysis were the murine anti-IGF-1Rantibodies αIR3 (Calbiochem, San Diego, Calif.) or MAB391 (R&D systems,Minneapolis, Minn.), 24-57 (Biocarta, San Diego, Calif.) and 1H7 (SantaCruz Biotechnology, Inc., Santa Cruz, Calif.). The negative controlantibody was an anti-CD20 antibody. Ligand competition data are shown inFIG. 15. The Ki and maximum inhibition values observed for IGF-1 andIGF-2 binding reactions are listed in Table 6.

TABLE 6 IGF-1 IGF-2 Antibody Ki (nM)¹ Max (%)² Ki (nM)¹ Max (%)² TQ11C0.6 84 0.3 91 TQ25 0.8 88 0.8 94 TQ58 0.8 91 0.8 91 TQ59 1.5 79 1.4 911H7 16.0 89 13.1 99 αIR3 5.3 91 No Inhibition ¹Ki of inhibition.²Maximum level of inhibition at 1 μM antibody concentration.

Example 10: SPA Dose Response Competition Assay

This example presents a scintillation proximity assay (SPA) forassessing the effect of antibodies on the interaction of insulin (INS)with the insulin receptor (INSR) and of IGF-1 and IGF-2 to IGF-1R.

IGF-1R binding reactions for TQ11C, TQ25, TQ 58 and TQ59 IgG1 antibodiescontained 1×PBS, 0.05% TWEEN® 20 (Mallinkrodt), 0.1% BSA (EM Science,Gibbstown, N.J.), 50 ng IGF-1R(ECD)-C3-muFc, 500 ug SPA PVT anti-mouseIgG fluoromicrospheres (Amersham) and ¹²⁵I-labeled IGF-1 or IGF-2obtained from Amersham at a final concentration of 0.64 nM. The totalreaction volume was 100 μl. The INSR binding reactions were identicalexcept they contained 50 ng INSR(ECD)-muFc and 0.64 nM ¹²⁵I-INS(Amersham). Receptor was loaded onto SPA PVT microspheres for 1 h atroom temperature prior to assembly of the binding reactions. To generatedose response data, antibodies or unlabeled growth factors were added atincreasing concentrations (10⁻¹¹ M to 10⁻⁶ M) simultaneously with¹²⁵I-labeled growth factors. Essentially all binding was competed withexcess unlabeled growth factors. The receptor-independent background,caused by random γ stimulation of the SPT PVT microspheres, was lessthan 0.5% of the input ¹²⁵I cpm. The data were expressed as the percentof total ligand bound minus background remaining after competition withexcess unlabeled growth IGF1 or IGF-2. Competition curves were generatedwith GraphPad Prism software using a single component equilibrium model.

Example 11: Antibody Binding to IGF-1R

This example provides a method of detecting the binding of ananti-IGF-1R antibody to IGF-1R.

BIACORE® 2000, sensor chip CMS, surfactant P20, HBS-EP (10 mM HEPES,0.15 M NaCl, 3.4 mM EDTA, 0.005% P20, pH 7.4), amine coupling kit, 10 mMacetate pH 4.5 and 10 mM glycine pH 1.5 all were purchased from BIACore,Inc. (Piscataway, N.J.). Phosphate-buffered saline (PBS, 1×, no calciumchloride, no magnesium chloride) was from Gibco. Bovine serum albumin(BSA, fraction V, IgG free) was from Sigma. Recombinant Protein G(“rProtein G”) was from Pierce Biotechnology.

Immobilization of rProtein G and IGF-1R-C3-muFc to the sensor chipsurface was performed according to manufacturer's instructions, using acontinuous flow of 10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.005% P20, pH7.4 (HBS-EP buffer). Briefly, carboxyl groups on the sensor chips'ssurfaces were activated by injecting 60 μl of a mixture containing 0.2 MN-ethyl-N′-(dimethylaminopropyl)carbodiimide (EDC) and 0.05 MN-hydroxysuccinimide (NHS). Specific surfaces were obtained by injectingrProtein A (Pierce) or IGF-1R-C3-mFc diluted in 10 mM acetate, pH 4.5 atconcentrations between 20 and 50 μg/ml. Excess reactive groups on thesurfaces were deactivated by injecting 60 μl of 1 M ethanolamine. Finalimmobilized levels were 5,000-6,000 resonance units (RU) for the ProteinG surfaces, and ˜7,800 RU for the IGF-1R-mFc surfaces. A blank,mock-coupled reference surface was also prepared on the IGF-1R-mFcsensor chip.

The kinetic analysis of the interaction between IGF-1R-mFc andantibodies was performed as follows. Antibodies as well as a positivecontrol antibody (anti-IR3-CDR-human-mouse chimera) were diluted inPBS+0.005% P20+0.1 mg/ml BSA and injected over the Protein G surfaces tocapture the antibodies. IGF-1R-mFc was diluted in PBS+0.005% P20+0.1mg/ml BSA from 500 nM to 3.9 nM, and each concentration was injectedover the captured antibody surfaces, as well as over a blank Protein Gsurface for background subtraction. After a 10 minute dissociation, eachsurface was regenerated by injecting 10 mM glycine, pH 1.5. Kineticanalysis of the resulting sensorgrams was performed using BIAEvaluation,v. 3.2 (BIACore, Inc.).

A solution affinity analysis was done by incubating two differentconcentrations (0.2 nM and 1 nM) of antibody with varying concentrations(0.01 nM to 50 nM) of IGF-1R-mFc in PBS+0.005% P-20+0.1 mg/ml BSA.Incubations were done at room temperature for at least five hours toallow samples to reach equilibrium. Samples were then injected over theimmobilized IGF-1R-mFc surface. After the sample injection, the surfaceswere regenerated by injecting 25 μl 8 mM glycine, pH 1.5. The bindingsignal obtained is proportional to the free antibody in solution atequilibrium. The dissociation equilibrium constant (K_(D)) was obtainedfrom nonlinear regression analysis of the competition curves using adual-curve one-site homogeneous binding model (KinExA software v. 2.3,Sapidyne Instruments Inc., Boise Id.). The data are shown in Table 7

TABLE 7 Kd (k_(a)/k_(d)) Kd Kinetic Equilibrium Antibody k_(oa) (1/Ms)K_(d) (1/s) Method Method TQ11C 6.0 × 10⁴ 6.7 × 10⁻⁵ 1.1 nM 0.3 nM TQ254.4 × 10⁴ <<5 × 10⁻⁵  0.10 nM TQ58 1.1 × 10⁵ 2.8 × 10⁻⁵ 0.25 nM 0.25 nMTQ59 6.9 × 10⁴ 2.1 × 10⁻⁴ 3.0 nM 0.30 nM

Example 12: Epitope Mapping Avidin-Fusion Proteins

This example provides a method of determining the epitope of IGF-1Rbound by an anti-IGF-1R antibody.

The subdomains of IGF-1R bound by antibodies TQ11C, TQ25, TQ58, and TQ59were determined using avidin-IGF-1R fusion proteins. To express eachprotein the coding DNA sequences of the complete IGF-1R(ECD) was clonedinto the expression vector pCep4-avidin-C such that chicken avidinsequence is joined to the C-terminus of the expressed IGF-1R protein.The ECD coding sequence (1-932) was PCR amplified from a parental IGF-1Rplasmid using PCR primers

2804-25: SEQ ID NO: 265 5′ GCAAGCTTGGGAGAAATCTGCGGGCCAG 3′ and 2826-68:SEQ ID NO: 266 5′ ATTGCGGCCGCTTCATATCCTGTTTTGGCCTG 3′

The primers include a 5′ Hind III site and a 3′ Not I site for cloninginto pCep4avidin-C. The amino acid sequence of the avidin-humanIGF-1R(ECD) fusion protein is shown in FIG. 12. The IGF-1R subdomainsconstructs used for epitope mapping included: L1 (1-151), CR (152-298),L2 (299-461), FnIII-1 (461-579), FnIII-2/ID (580-798), FnIII-3(799-901), L1+CR+L2 (1-461), and L1+CR (1-298). The amino acidcoordinates of the IGF-1R subdomain represented in each expressionplasmid are given in parenthesis. The coding sequence of each domain wasPCR amplified from a parental IGF1R cDNA clone using the followingprimer pairs:

L1: 2804-25: (SEQ ID NO: 265) 2804-19: SEQ ID NO: 267 5′ATTGCGGCCGCCCCACATTCCTTTGGGGGC 3′ CR: 2804-38: SEQ ID NO: 268 5′AGCAAGCTTGGACCTGTGTCCAGGGACC 3′ 2804-20: SEQ ID NO: 269 5′ATTGCGGCCGCGCAAGGACCTTCACAAGGG 3′ L2: 2804-39: SEQ ID NO: 270 5′AGCAAGCTTGCCGAAGGTCTGTGAGGAAG 3′ 2804-23: SEQ ID NO: 271 5′ATTGCGGCCGCACTTTCACAGGAGGCTCTC 3′ FnIII-1: 2808-08: SEQ ID NO: 272 5′AGCAAGCTTGGACGTCCTGCATTTCACCTC 3′ 2804-52: SEQ ID NO: 273 5′ATTGCGGCCGCGGTGCGAATGTACAAGATCTC 3′ FnIII-2 + ID: 2804-41:SEQ ID NO: 274 5′ AGCAAGCTTGAATGCTTCAGTTCCTTCCATTC 3′ 2804-51:SEQ ID NO: 275 5′ ATTGCGGCCGCAGTCCTTGCAAAGACGAAGTTG 3′ FnIII-3: 2804-42:SEQ ID NO: 276 5′ AGCAAGCTTGATGCCCGCAGAAGGAGCAG 3′ 2804-50:SEQ ID NO: 277 5′ ATTGCGGCCGCTTTAATGGCCACTCTGGTTTC 3′ L1 + CR + L2:2804-25: SEQ ID NO: 278 5′ AGCAAGCTTGGGAGAAATCTGCGGGCCAG 3′ 2804-23(SEQ ID NO: 272) L1 + CR: 2804-25: (SEQ ID NO: 279)AGC AAG CTT GGG AGA AAT CTG CGG GCC AG 2804-20 (SEQ ID NO: 270)

The primers included Hind III and Not I site for cloning as describedfor the IGF-1R (ECD). The IGF-1R subdomains were cloned into theexpression vector pCep4avidin-N such that chicken avidin sequence (withendogenous signal sequence) is joined to the N-terminus of the expressedIGF-1R proteins.

Expression of each avidin-fusion protein was achieved by transienttransfection of human 293-EBNA cells (Invitrogen) in roller bottlescultures. The cells were grown and maintained in DMEM supplemented with5% FBS+1× Non-Essential Amino Acids+1× Pen Strep Glut+1× SodiumPyruvate. Approximately 4-5×10⁷ 293-EBNA cells were seeded in 850 cm²roller bottles overnight. The previously seeded cells were thentransfected with pCep4-avidin plasmid DNA the following day usingFUGENE™ 6 transfection reagent. The DNA-transfection reagent mixture wasprepared in approximately in 6.75 mL serum-free DMEM. 675 μl FUGENE™ 6transfection reagent was first added, followed by 112.5 μg plasmid DNA.The complex was incubated at room temperature for 30 minutes. The entiremixture was then added to a roller bottle. The roller bottle was gassedwith a 5% CO₂ gas mixture, capped tightly and placed in a 37° C.incubator on a roller rack rotating at 0.35 RPM. The transfection wasperformed for 24 hours after which the medium was replaced with 100 mLDMEM+1× Insulin-Transferrin-Selenium Supplement+1× Pen Strep Glu+1×Non-Essential Amino Acids+1× Sodium Pyruvate. Harvest of the conditionmedium and replacement with fresh medium occurred 48 hr intervals (2-3cycles). The harvested serum-free conditioned medium was pooled togetherand clarified by centrifugation at 10,000×g for 30 minutes at 4° C.

The concentration of avidin-fusion in each conditioned medium wasdetermined using a quantitative FACS based method. The avidin fusionprotein in 200 μl of conditioned medium was captured by incubation for 2hr at room temperature with 5 μl (˜3.5×10⁵) of biotin coated polystyrenebeads (Spherotech, Inc., Libertyville, Ill.). The conditioned medium wasremoved by three cycles of centrifugation and resuspension of theavidin-coated beads in PBS containing 0.5% BSA (BPBS). The avidin-beadswere stained with 1 μg/ml of goat FITC-labeled anti-avidin antibody(Vector Lab Burlingame, Calif.) in 1 ml BPBS. After 0.5 hr incubationantibody-beads complexes were collected by centrifugation at 1800 rpmfor 5 min and the pellet was washed three times. The FITC fluorescencewas detected with a FACSCAN (Beckton Dickson Bioscience, Franklin Lakes,N.J.). The signal was converted to protein mass using a standard curvederived with recombinant avidin. For epitope mapping the biotin-beadswere loaded with 50-100 ng avidin-fusion protein per ˜3.5×10⁵ beads ofbeads by incubation with the appropriate amount (1-20 ml) of conditionedmedium. The loaded beads were washed extensively and resuspended in lmlBPBS. For all experiment the biotin-beads were blocked with 10% BSA inPBS prior to loading fusion protein.

Method 1, One Color Assay:

Biotin-coated polystyrene beads loaded with IGF-1R (ECD) and IGF-1Rsubdomain fusion proteins were mixed with 1 μg of anti-IGF-1R antibodyin 1 ml of BPBS. After incubation for 1 hr at room temperature, 4 mlwashing buffer was added and the antibody-beads complexes were collectedby centrifugation for 5 min at 750 g. The pellet was washed 3 times byresuspension in 4 ml of BPBS. The antibody bound to avidin-beadcomplexes was detected by treatment with 0.5 μg/ml Phycoerythrin-(PE)labeled goat anti-human F(ab′)2 (Southern Biotech Associates, Inc.,Birmingham, Ala.) in 1 ml BPBS. Tested antibodies were found to bind tothe avidin-fusion protein containing the complete IGF-1R ECD and the L2domain. Binding to L1, CR or FnIII-1 was not detected in thisexperiment. A relatively weak reaction was also observed with the L1domain.

Method 2, Two Color Assay:

To simultaneously monitor the amounts of anti-IGF-1R monoclonal antibodyand avidin-fusion bound to biotin-beads, FITC-labeled anti-avidinantibody was included (1 μg/ml) was included in the binding reaction incombination with 0.5 μg/ml PE-labeled goat anti-human IgG1. The beadswere prepared for FACSCAN analysis as described for the one color assay.

Method 3, Antibody Competition:

To prepare for labeling with fluorescein the antibodies were dialyzed orresuspended at a concentration of 1 mg/ml in PBS (pH 8.5). Label([6-fluorescein-5-(and-6)-carboxamido]hexanoic acid, succinimidyl ester5(6)-SFX] mixed isomers from Molecular Probes (Eugene, Oreg., Cat. No.F2181) was added to the protein at a molar ratio 9.5:1 (label: protein)from a label stock of 5 mg/ml in DMSO. The mixture was incubated at 4°C. overnight in the dark. The labeled antibody was separated from thefree label by dialysis in PBS. The FITC/antibody ratios obtained rangedfrom 3 to 8. For each competition experiment, a binding reaction wasassembled that contained a 50 fold excess (10-50 μg/ml) of unlabeledcompetitor antibody, 3.5×10⁵ biotin beads coated with avidin fusionprotein in BPBS. The FITC-labeled antibody (1 μg/ml) was added after a30 min preincubation. The process followed the one color method fromthis point forward.

Each of the four tested antibodies binds to the IGF-1R L2 domain, asshown in Table 8. However, the precise amino acid contacts of eachantibody in the IGF-1R L2 domain may differ.

TABLE 8 Antibody L1¹ CR¹ L2¹ FnIII-1¹ ECD^(1,2) TQ11C No No Yes No YesTQ25 No No Yes No Yes TQ58 Yes No Yes No Yes TQ59 No No Yes No Yes¹Epitope mapping was performed with avidin-IGF-1R fusion proteinscontaining the indicated human IGF-1R regions. ²The BCD fusion containsL1 + CR + L2 + FnIII-1 + FnIII-2 + ID + FnIII-3.

Example 13: Antibody Binding to Cell-Surface IGF-1R

This example provides a method for detecting the binding of ananti-IGF-1R antibody to cell-surface expressed IGF-1R.

The ability of antibodies TQ11C, TQ25, TQ58, and TQ59 to bind to humanIGF-1R displayed on the cell surface was evaluated using Balb/C 3T3fibroblasts and MCF-7 human breast cancer cells engineered tooverexpress the human IGF-1R receptor at a level of −3-4×10⁵ moleculesper cell. A Balb/C 3T3 cell line that stably overexpresses the humanIGF-1R (˜3×10⁵ receptors per cell) was derived using with a retroviralvector essentially as described by Pietrzkowski et al., 1992, CellGrowth Differentiation 3:199-205. MCF-7 breast cancer cells thatoverproduce huIGF-1R were transfected with a pcDNA3.1 expression vector(Invitrogen Corp.). Zeocin resistant cells that express a high level ofhu IGF-1R (˜4×10⁵ receptors per cell) were expanded after selection byFACS using anti-IGF-1R monoclonal antibody αIR3 and an PE-labeled goatanti murine IgG antibody (Caltag Laboratories, Burlingame, Calif.). Theprocess of selection and expansion was repeated four times.

IGF-1R Receptor antibody staining and receptor expression was monitoredby FACS as follows: the cells were released from T175 flasks (Corning)by washing 2 times with excess PBS (Ca/Mg free) followed by treatmentwith 5 ml of Cell Dissociation Buffer (Sigma) for 10 min at roomtemperature. The cells were collected by centrifugation and washed twotimes by resuspending them in PBS and centrifugation. For primaryantibody staining, 1 μg of antibody was added to 10⁶ cells resuspendedin 100 μl PBS plus 0.5% BSA (BPBS) and the cells were incubated at 4° C.for 1.5 hr. The cells were collected by centrifugation and washed twicewith BPBS to remove unbound primary antibody. The cells were resuspendedin 100 μl of BPBS and incubated with 1 μg of FITC-labeled goatanti-human F(ab′)2 (Southern Biotechnology Associates, Inc., Birmingham,Ala.) at 4° C. for 30 minutes. After washing to remove unbound FITCsecondary antibody, the cells were resuspended in 1 ml of PBS+0.5% BSAand FITC cell fluorescence was detected with a FACSCAN (Beckton DicksonBioscience, Franklin Lakes, N.J.). The fluorescence levels wereconverted to absolute receptor levels using Quantum microbead (BangsLaboratories, Inc., Fishers, Ind.) with predetermined IgG1 bindingcapacity to generate a standard curve. Data reduction was performed withQuickCal v2.1 software (Verity Software House, Topsham, Me.) provided bythe manufacturer.

The peak fluorescent intensity of anti-IGF-1R antibody labeling of theIGF-1R overexpressors was increased 10-20 fold relative to parentalBalb/C 3T3 and MCF-7 cells for each of the tested antibodies. This isthe result predicted for an antibody that specifically binds IGF-1R.Background fluorescence of cells treated with no antibodies orFITC-labeled secondary alone were insignificant.

Example 14: Inhibition of IGF-1R

This example presents methods of detecting inhibition of IGF-1R byanti-IGF-1R antibodies.

32D hu IGF-1R+IRS-1 Cell Inhibition

Murine 32D cells that coexpress the human IGF-1R receptor (20K per cell)and human IRS-1 have proven to be a effective system to examine themolecular components IGF-1R signaling Valentinis et al., 1999, J BiolChem 274:12423-30. Normal 32D cells express relatively low levels of themurine orthologs of these two gene products. 32D cell normally requiredIL3 for growth and survival. IGF-1 or IGF-2 can replace IL3 in 32DhuIGF-1R+IRS-1 cells as shown in FIG. 16, panel A. The EC₅₀ to the IGF-1dose response curve was about 0.5 nM, whereas the IGF-2 EC₅₀ (2.8 nM) isabout six fold higher reflecting weaker affinity of IGF-2 for IGF-1R. Toassess the ability of the antibodies TQ11C, TQ25, TQ58, and TQ59 toblock IGF-1 or IGF-2 stimulation, 96-well microtitre plates were seededwith 30,000 32D hu IGF-1R+IRS-1 cells per well in a volume of 200 μl ofRPMI (Gibco/BRL) containing 5% fetal bovine serum (Gibco/BRL) and 1×penicillin, streptomycin, glutamine (Giboco/BRL) and increasingconcentrations of antibody (10⁻¹² M to 10⁻⁶ M) or no antibody. IGF-1 (2nM), IGF-2 (8 nM) or nothing was added after 1 hr preincubation withantibody. ³H-thymidine (1 μCi per well) was added at 27 hr post-antibodyaddition. The cells were harvested 21 hr later, and incorporation of³H-thymidine into DNA was determined for each sample. The assays wereperformed in triplicate. An anti-CD20 antibody was used as a negativecontrol. Each of antibodies TQ11C, TQ25, TQ58, and TQ59 was able tocompletely block the IGF-1 and IGF-2 mediated stimulation of the 32Dcells. The reduction of background proliferation in the absence of addedIGF-1 and IGF-2 is due to the inhibition of serum IGF-1 and IGF-2. Thebinding data were analyzed using GraphPad PRIZM™ software. The data areshown in FIG. 16.

Balb/C 3T3 hu IGF-1R Cell Inhibition

IGF-1 greatly stimulates the incorporation of ³H-thymidine byserum-starved cultures of mouse embryonic fibroblasts (Balb/C 3T3 or NIH3T3) that overexpress IGF-1R (˜1×10⁶ IGF1R per cell). Kato et al., 1993,J Biol Chem 268:2655-61; Pietrzkowski et al., 1992, Cell GrowthDifferentiation 3:199-205. This phenomenon is recapitulated with bothIGF-1 and IGF-2 in a Balb/C 3T3 cell line hu IGF-1R overexpressor. Bothgrowth factors stimulated ³H-thymidine incorporation by about 20-fold.The EC₅₀ of the IGF-1 dose response curve was about 0.7 nM, whereas theIGF-2 EC₅₀ (4.4 nM) is sevenfold higher, indicating a weaker affinity ofIGF-2 for IGF-1R. To assess the ability of a given antibody to blockIGF-1 or IGF-2 stimulation, 96-well microtitre plates were seeded with10,000 cells per well in a volume of 200 μl of DMEM (Gibco/BRL)containing 10% calf serum (Gibco/BRL) and 1× penicillin, streptomycin,glutamine (Giboco/BRL). After overnight incubation when the cells wereabout 80% confluent the growth medium was replaced with 100 μl DMEMcontaining 0.1% BSA after washing once with 200 μl PBS. Antibodies atincreasing concentrations (10⁻¹² M to 10⁻⁶ M), or no antibody, wereadded at 24 hr post-serum starvation. IGF-1 (2 nM), IGF-2 (8 nM) and³H-thymindine (1 μCi per well) were added after a 1 hr preincubationwith antibody. The cells were harvested 24 hr later, and incorporationof ³H-thymidine into DNA was determined for each sample. The assays wereperformed in triplicate. Each tested antibody was able to completelyblock the IGF-1 and IGF-2 mediated stimulation of Balb/C 3T3 cells, asshown in FIG. 17. An anti-CD20 antibody was used as a negative control(“CD20” in FIG. 17).

Each reference cited herein is incorporated by reference in its entiretyfor all that it teaches and for all purposes.

What is claimed is:
 1. An isolated anti-IGF-1R antibody comprising alight chain variable region and a heavy chain variable region, whereinsaid light chain variable region is at least 90 percent identical to SEQID NO:32 and said heavy chain variable region is at least 90 percentidentical to SEQ ID NO:136, and wherein said light chain variable regioncomprises: i. the CDR 1 sequence of residues 24 through 39 of SEQ IDNO:32; and ii. the CDR 2 sequence of residues 55 through 61 of SEQ IDNO:32; and iii. the CDR 3 sequence of residues 94 through 102 of SEQ IDNO:32; and said heavy chain variable region comprises: i. the CDR 1sequence of residues 31 through 36 of SEQ ID NO:136; and ii. the CDR 2sequence of residues 51 through 66 of SEQ ID NO:136; and iii. the CDR 3sequence of residues 99 through 108 of SEQ ID NO:136.
 2. The isolatedanti-IGF-1R antibody of claim 1, that, when bound to IGF-1R: a. inhibitsIGF-1R.
 3. The anti-IGF-1R antibody of claim 1, that, when bound to ahuman IGF-1R, inhibits binding of IGF-1 and/or IGF-2 to said humanIGF-1R.
 4. The anti-IGF-1R antibody of claim 1, that binds to humanIGF-1R with a selectivity that is at least fifty times greater than itsselectivity for human insulin receptor.
 5. The anti-IGF-1R antibody ofclaim 1, that inhibits tumor growth in vivo.
 6. The anti-IGF-1R antibodyof claim 1, that inhibits IGF-1R mediated tyrosine phosphorylation.
 7. Apharmaceutical composition comprising the antibody of claim 1 and apharmaceutically acceptable carrier, diluent, or excipient.